Novel Method for Obtaining Efficient Viral Vector-Based Compositions for Vaccination or Gene Therapy

ABSTRACT

The present invention relates to a method for preparing viral vector-based compositions wherein the viral vector-based particles present in the composition have a particle size distribution with a polydispersity index (PDI) of less than 0.5, the method comprising the steps: (a) providing replication-deficient viral vectors; (b) providing a solution comprising at least one sugar and at least three different excipients selected from hydrophilic and amphiphilic excipients, wherein the excipients are characterized by polar, aliphatic, aromatic, negatively charged, and/or positively charged functional groups, and wherein the solution is further characterized by an excipient-sugar ratio of at least 1:2 (w/w); and (c) mixing the replication deficient viral vectors of step (a) with the solution of step (b). The present invention further relates to a viral vector-based composition obtainable by the method of the invention as well as to the viral vector-based composition of the invention for use as a prime-boost vaccine.

The present invention relates to a method for preparing viralvector-based compositions, wherein the viral vector-based particlespresent in the composition have a particle size distribution with apolydispersity index (PDI) of less than 0.5, the method comprising thesteps: (a) providing replication-deficient viral vectors; (b) providinga solution comprising at least one sugar and at least three differentexcipients selected from hydrophilic and amphiphilic excipients, whereinthe excipients are characterized by polar, aliphatic, aromatic,negatively charged, and/or positively charged functional groups, andwherein the solution is further characterized by an excipient-sugarratio of at least 1:2; and (c) mixing the replication deficient viralvectors of step (a) with the solution of step (b). The present inventionfurther relates to a viral vector-based composition obtainable by themethod of the invention as well as to the viral vector-based compositionof the invention for use as a prime-boost vaccine.

In this specification, a number of documents including patentapplications and manufacturer's manuals are cited. The disclosure ofthese documents, while not considered relevant for the patentability ofthis invention, is herewith incorporated by reference in its entirety.More specifically, all referenced documents are incorporated byreference to the same extent as if each individual document wasspecifically and individually indicated to be incorporated by reference.

Replication-deficient recombinant viral vectors represent a rapidlygrowing field of vaccine development and gene therapy. When intended foruse in vaccination, viral vectors and virus like particles (VLPs) offera series of advantages over traditional vaccines. In addition toinducing exceptional antibody responses, they also elicit cytotoxic Tlymphocytes (CTL) that are crucial for the control of intracellularpathogens and cancer, a feature not observed by protein-based vaccines[Rollier CS et al., 2011]. Many viral species have been evaluated asrecombinant vectors for vaccines, including retrovirus, lentivirus,vaccinia virus (e.g. modified vaccinia Ankara virus; MVA), adenovirus,adeno-associated virus, cytomegalovirus, Sendai virus, measles virus andvesicular stomatitis virus (VSV). However, the most widely evaluatedvectors to date are adenovirus type 5 and members of the poxvirus family[Rollier CS et al., 2011, Ura T et al.2014].

A drawback associated with viral vectors, in particular uponmanufacturing, storage and distribution, is that they are complexsupra-molecular ensembles of macromolecules which are prone to a varietyof chemical and physical degradation pathways [Vrdoljak A et al. 2012].Thus, a major challenge in this field is the reduction (avoidance) ofcross-linking and vector particle interaction of neighboring virusparticles that is typically caused over a broad range of concentrationsby various mechanisms at different stages of production, storage andapplication. This intrinsic tendency of viral vectors for particleagglomeration of different shapes and sizes within a composition leadsto inhomogeneous size distribution of the viral particles and anassociated increase in polydispersity. Ultimately, these effects resultin a significant loss of therapeutic efficacy, and can even lead toadverse effects at the injection site, most likely due to the increasedviscosity observed as a result of said particle agglomeration.Furthermore, aggregation of the viral vectors is also considered toinfluence biodistribution after administration and, similar to proteinpharmaceuticals, aggregation of viral vectors may increase undesiredimmunogenicity by targeting the vector to antigen presenting cells,thereby inducing or enhancing undesired immune responses to the surfaceproteins or protein capsids and transgenic products. As highpolydispersity is associated with high viscosity, compositions that donot show such unappreciated polydispersity are expected to also lead tobetter syringeability and injectability. Thus, improved viralvector-based vaccines with low polydispersity and having a more suitableratio between vector particle distribution and functional efficacy wouldbe highly desired.

Similar considerations apply when viral vectors are intended for use asgene transfer therapeutics. Viral vectors have emerged as safe andeffective delivery vehicles for clinical gene therapy, as shown in aseries of clinical studies, especially for monogenic recessivedisorders, but also for some idiopathic diseases (see e.g. Kotterman MAet al., Viral Vectors for Gene Therapy: Translational and ClinicalOutlook. Annu Rev Biomed Eng. 2015 Dec 7;17:63-89). These clinicalstudies were conducted on the basis of vectors that combine lowgenotoxicity and immunogenicity with highly efficient delivery,including vehicles based on adeno-associated virus and lentivirus, whichare increasingly enabling clinical success. Important examples forclinical treatment strategies based on viral vectors include, e.g., stemcell therapy, mucoviscidosis, haemophilia, inherited retinopathy orcystic fibrosis. (Collins M, ThrasherA, Gene therapy: progress andpredictions. Proc Biol Sci. 2015;282). Typically, the viral vectorsemployed in such gene transfer therapeutics include retrovirus,adenovirus, adeno-associated virus (AAV) and herpes simplex virus.

Also with regard to gene transfer therapeutics, the avoidance ofunappreciated polydispersity for obtaining a more suitable ratio betweenvector particle distribution and functional efficacy of gene transfervectors is essential for efficient host cell infection and subsequentgene expression. In particular, the in vivo administration of genetherapeutic viral vectors to certain sites, such as the central nervoussystem, is expected to require small volumes of highly concentratedviral vectors, a feature for which the maximum achievable dose may belimited by the intrinsic property of low vector solubility. Thus, atpresent, there are still substantial delivery challenges that have to beovercome to extend the success achieved so far to a broad variety ofdiseases; these challenges include developing techniques to evadepre-existing immunity, to ensure more efficient transduction oftherapeutically relevant cell types, to target delivery, and to ensuregenomic maintenance.

Formulation development for virus-based viral vector compositions forvaccines or gene-transfer therapeutics is rather difficult, mainly dueto their complex molecular structure. Thus, formulation development forviral vector based pharmaceutical compositions is a relatively recentarea of investigation and only a few studies and patent applicationshave been reported describing systematic efforts to optimize viralvector formulations and stability. An important aspect of vectorstability is solubility during vector purification, preparation andstorage.

U.S. Pat. No. 7,704,721 describes compositions and methods to preventthe aggregation of recombinant adeno associate virus (AAV) virionspurified using ultracentrifugation and/or chromatography by adding oneor more salts of multivalent ions in high concentrations to produce apreparation of the virions that are nonetheless isotonic with theintended target tissue. This combination of high ionic strength andmodest osmolarity is achieved using salts of high valency, such assodium citrate. High concentrated AAV stock formulations for genetherapy with up to 6.5×10¹³ viral particles per ml can be prepared andstored in this way without aggregation. No aggregation was observed bydynamic light scattering (DSL) even after 10 freeze/thaw cycles. Thesurfactant Pluronic F68 may be added at 0.001% to prevent losses ofvirions to surfaces during handling. Virion preparations can also betreated with nucleases to eliminate small nucleic acid strands on virionsurfaces that exacerbate aggregation. These highly specific formulationsfor AAV compositions described in U.S. Pat. No. 7,704,721 are purposelyderived from specific intrinsic structural conditions of the AAV virionsand require the presence of high amounts of multivalent ions andsurfactants as well as an additional nuclease treatment. Thus, theseformulations cannot simply be transferred generically to otherviral-vector based compositions having a wide range of differentproperties. Instead, individual adjustments for each new viral-vectorbased composition are required, depending on the respective individualproperties.

WO 2009022174 describes non aggregating virus formulations comprising avirus, preferably an adenovirus, a polyol, preferably glycerol, and azwitterionic compound, preferably HEPES. Additionally, an assay forviral aggregation using dynamic light scattering (DLS) is describedwhich comprises analyzing the size of viral particles in a samplewherein the particles are in a mixture with polyol and determining fromthe size whether the sample contains substantially only acceptablenon-aggregated particles after repeated freeze and thaw cycles andstorage of the compositions at ambient temperature. It is concluded inthe document that the composition can be utilized with a wide range ofviruses, preferably adenovirus. Although WO 2009022174 describes anon-aggregating virus formulation, said formulations necessarilycomprise glycerol and a buffer like the disclosed zwitterionic bufferingsubstance HEPES. However, using a polyol such as glycerol has thedrawback of being associated with an increase in unappreciatedviscosity, which in turn results in unfavorable syringeability andinjectability of a vaccine. The use of the buffering substance HEPES hasthe additional drawback of the possible appearance of phototoxic effectsas a result of the exposure to ambient light and the subsequentformation of hydrogen peroxide.

U.S. Pat. No. 7,888,096 describes liquid and lyophilized adenovirusformulations with improved long term storage stability at 4° C., interms of infectivity. The liquid and lyophilized formulations areprepared for use in gene therapy, commonly using retroviruses,adenoviruses, or lentiviruses. The authors of U.S. Pat. No. 7,888,096emphasize that the particles must maintain their biological integrity tobe infectious. They further describe for dry formulations the use of abulking agent for lyophilization (mannitol), a cryoprotectant (sucrose),a lyoprotectant (human serum albumin), several buffers, and salts. Theliquid formulation described in U.S. Pat. No. 7,888,096 comprisesbuffers and polyol and an additional nuclease treatment is requiredduring purification in order to prevent aggregation. As discussed above,polyols may increase unappreciated viscosity in the vaccine product.However, nucleases have the drawback that they may exert unappreciatedeffects in vivo.

WO 2015040234 describes pharmaceutical adenovirus formulations for usein gene therapy as well as in vaccines, in particular liquidpharmaceutical formulations comprising adenovirus, a histidine bufferedsolution, trehalose, a salt and a non-ionic detergent, wherein the pH isranging between 6-7. The resulting formulation has been shown topreserve the quantity, potency (infectivity) and quality of thecontaining adenoviruses, therewith improving the overall adenoviralstability compared to other formulations known in the art. Theadenoviral formulations according to WO 2015040234 are amenable toprolonged storage at 2 to 8° C. for more than 6 months comprising theadenovirus at a titer ranging between 1×10⁷ and 1×10¹³ virus particlesper milliliter. However, the analysis of the recited storage stabilityof the adenoviral formulations is only based on the combination of aqPCR analysis and a cell culture based infectivity assay. Thermalmelting assays using Dynamic light scattering (DLS) analysis were usedto analyze the melting temperature of the adenoviral capsid withincreasing temperature. The accompanying changes in the polydispersityindex were, however, not monitored. Furthermore, changes inpolydispersity indices upon purification and storage were also notdetermined. However, although several physical and chemicalinstabilities of adenoviral vectors are summarized (e.g. aggregation,deamidation, oxidation, degradation etc.) and the resulting challenges,especially in the stabilization and formulation development ofadenoviral vectors, are discussed, the instabilities cited in WO2015040234 are rather typical for proteins. Thus, the describedstabilizing formulations do not address the more complex VLPs and/orviral vectors and their tendency to aggregate with the consequence ofhigher polydispersity in a suspension.

US20100124557 describes a liquid or liquid-frozen composition comprisinga modified vaccinia Ankara (MVA) virus or derivative thereof andmannitol as the sole stabilizing agent of the composition. Mannitol hasbeen shown to exert the stabilizing effect at temperatures between 0° C.to 10° C. in the liquid state and in a liquid-frozen state attemperatures between −10 to −30° C. The storage stability of the MVAvirus in the mannitol formulations was only analyzed in cell culturebased infectivity assays. However, no changes in the particle sizedistribution profiles and in the corresponding polydispersity indicesupon preparation and storage resulting in loss of function in terms ofinfectivity were analyzed.

WO 2013001034 describes the utilization of viruses for the generation ofviral or bacterial antigens. The stabilization of viral surfacemolecules is described, thereby enabling a prolonged storage andinfectivity of replication competent viruses. In addition, it isdescribed that the antigenicity of proteins is maintained even afterirradiation, if the composition comprised amino acids. However, theproblem of an increasing polydispersity of non-replicating VLPs or viralvectors in suspensions is not addressed.

All these approaches have in common that the stability of viruses orviral proteins is addressed. For example, maintaining the molecularintegrity of viral proteins in replication competent viruses, such asligands for cellular target molecules, is important for cellularinfection. Moreover, the antigenicity of relevant viral proteins is ofinterest in vaccine development. However, aside from these biologicalfunctions, the physical characteristics of particles within a suspensionrepresent an important aspect in vaccine production based on VLP and/orviral vectors. The problem of avoiding an increase in polydispersity ofa composition comprising VLPs and/or viral vectors and, by this, thedecreased vaccination or gene transfer efficacy, has not been solved sofar.

Thus, despite the fact that a lot of effort is currently being investedinto the development of novel vaccines or gene transfer therapeutics,there is still a need to provide improved compositions characterized bylow and medium polydispersity indices and, thus, a higher ratio betweeninfective particles and non-infective large particles or particleagglomerates within the composition. As a consequence of reducingpolydispersity, the efficacy of the vaccine or gene transfer approachcould be increased, thus resulting in a reduction of costs, as well as areduction of adverse events at the injection site and in the body.

This need is addressed by the provision of the embodiments characterizedin the claims.

Accordingly, the present invention relates to a method for preparingviral vector-based compositions wherein the viral vector-based particlespresent in the composition have a particle size distribution with apolydispersity index (PDI) of less than 0.5, the method comprising thesteps: (a) providing replication-deficient viral vectors; (b) providinga solution comprising at least one sugar and at least three differentexcipients selected from hydrophilic and amphiphilic excipients, whereinthe excipients are characterized by polar, aliphatic, aromatic,negatively charged, and/or positively charged functional groups, andwherein the solution is further characterized by an excipient-sugarratio of at least 1:2 (w/w); and (c) mixing the replication deficientviral vectors of step (a) with the solution of step (b).

Viral vectors are commonly used to deliver genetic material into cellsin vivo or in vitro. Viruses may efficiently transport their genomesinside the host cells. Virus-like particles resemble viruses, arenon-infectious and do not contain viral genetic material. The expressionof viral structural proteins, such as Envelope or Capsid, can result inthe self-assembly of virus like particles (VLPs). VLPs derived from theHepatitis B virus may be composed of the HBV surface antigen (HBsAg)(Hyakumura M. et al. J. Virol. 89:11312-22, 2015) or from HBV core(Sominskaya I. et al. PLos One 8:e75938). VLPs have been produced fromcomponents of various virus families including Parvoviridae (e.g.adeno-associated virus), Retroviridae (e.g. HIV), Flaviviridae (e.g.Hepatitis C virus) and bacteriophages (e.g. Qβ, AP205). VLPs can beproduced in different cell culture systems including bacterial,mammalian, insect, yeast and plant cell lines. The term “viralvector-based composition” as used herein, relates to a composition thatcomprises at least a viral vector. The term “viral vector”, inaccordance with the present invention, relates to a carrier, i.e. a“vector” that is derived from a virus. “Viral vectors” in accordancewith the present invention include vectors derived from naturallyoccurring or modified viruses, as well as virus like particles (VLPs).

In general, the starting materials for the development of viral vectorsare live viruses. Thus, certain requirements such as safety andspecificity need to be fulfilled in order to ensure their suitabilityfor use in animals or in human patients. One important aspect is theavoidance of uncontrolled replication of the viral vector. This isusually achieved by the deletion of a part of the viral genome criticalfor viral replication. Such a virus can infect target cells withoutsubsequent production of new virions. Moreover, the viral vector shouldhave no effect or only a minimal effect on the physiology of the targetcell and rearrangement of the viral vector genome should not occur. Suchviral vectors derived from naturally occurring or modified viruses arewell known in the art and have been described, e.g. in the Review ofLukashev A N and Zamyatnin A A “Viral Vectors for Gene Therapy: CurrentState and Clinical Perspectives”. Front Mol Neurosci. 2016;9:56 as wellas in the Review of Stoica L and Sena-Esteves M “Adeno Associated ViralVector Delivered RNAi for Gene Therapy of SOD1 Amyotrophic LateralSclerosis”, Front Mol Neurosci. 2016 Aug. 2;9:56.

Also vectors derived from virus like particles are well known in the artand have been described, e.g. in Tegerstedt et al. (Tegerstedt et al.(2005), Murine polyomavirus virus-like particles (VLPs) as vectors forgene and immune therapy and vaccines against viral infections andcancer. Anticancer Res. 25(4):2601-8.). One major advantage of VLPs isthat they are not associated with any risk of reassembly as is possiblewhen live attenuated viruses are used as viral vectors and, as such,they represent “replication-deficient viral vectors” in accordance withthe present invention. VLP production has the additional advantage thatit can be started earlier than production of traditional vaccines oncethe genetic sequence of a particular virus strain of interest has becomeavailable.

VLPs contain repetitive high density displays of viral surface proteinswhich present conformational viral epitopes that can elicit strong Tcell and B cell immune responses. VLPs have already been used to developFDA approved vaccines for Hepatitis B and human papillomavirus and,moreover, VLPs have been used to develop a pre-clinical vaccine againstchikungunya virus. Evidence further suggests that VLP vaccines againstinfluenza virus might be superior in protection against flu viruses overother vaccines. In early clinical trials, VLP vaccines for influenzaappeared to provide complete protection against both the Influenza Avirus subtype H5N1 and the 1918 flu as reviewed by Quan F S et al.,“Progress in developing virus-like particle influenza vaccines”.ExpertRev Vaccines. 2016 May 5:1-13.

Highly purified and homogenous VLPs can be formulated as so-called“lipoparticles”, which contain high concentrations of a conformationallyintact membrane protein of interest. Integral membrane proteins areinvolved in diverse biological functions and are targeted by nearly 50%of existing therapeutic drugs. However, because of their hydrophobicdomains, membrane proteins are difficult to manipulate outside of livingcells. Lipoparticles can incorporate a wide variety of structurallyintact membrane proteins, including G protein-coupled receptors (GPCR)s,ion channels, and viral envelopes. Lipoparticles may be used as platformfor numerous applications including antibody screening, production ofimmunogens, and ligand binding assays.

Virus-like particles can also be used as drug delivery vectors(Zdanowicz M and Chroboczek J, 2016).

The presence of viral structural proteins, for example, structuralproteins in the envelope or in the capsid, can result in theself-assembly of VLPs. In general, VLPs can be produced in a variety ofcell culture systems including mammalian cell lines, insect cell lines,yeast, and plant cells and VLPs have been produced from different virusfamilies including parvoviridae (e.g. adeno-associated virus),retroviridae (e.g. HIV), and flaviviridae (e.g. Hepatitis C virus). Forexample, VLPs derived from the Hepatitis B virus and composed of thesmall HBV-derived surface antigen (HBsAg) have been described bySominskaya I et al. Construction and immunological evaluation ofmultivalent hepatitis B virus (HBV) core virus-like particles carryingHBV and HCV epitopes. Clin Vaccine Immunol. 2010 Jun;17:1027-33.

In accordance with the present invention, the term “viral vectors”includes, without being limiting, (i) viral vectors represented by oneparticular type of viral vector, or (ii) viral vector mixtures ofdifferent molecular types of viral vectors.

The composition may, optionally, comprise further molecules capable ofaltering the characteristics of the viral vector(s). For example, suchfurther molecules can serve to stabilize, modulate and/or enhance thefunction of the viral vector(s). The viral vector-based compositionsprepared by the method of the present invention may be in solid orliquid form and may be, inter alia, in the form of (a) powder(s), (a)tablet(s) or (a) solution(s).

The viral vector-based composition prepared in accordance with thepresent invention is further characterized in that the particlescomprised in the composition have a particle size distribution with apolydispersity index (PDI) of less than 0.5.

The term “particle(s)”, as used herein, relates to the viral vector(s)that represent the main, active ingredient of the composition preparedin accordance with the invention. The term “particle size distribution”,in accordance with the present invention, refers to the relative amountof particles present according to size. Typically, the relative amountis determined by mass.

In accordance with the present invention, the particle size distributionis expressed in terms of the polydispersity index (PDI). Polydispersityand the polydispersity index are parameters measured by Dynamic LightScattering (DLS) and characterize a dispersion or solution in additionto the typically determined main parameters, i.e. particle size andhydrodynamic diameter of particles. DLS measures time-dependentfluctuations in the scattering intensity arising from particles, such ase.g. viral particles or proteins undergoing random Brownian motions(diffusion). A monochromatic light beam, such as a laser beam, causes aDoppler shift in a solution with particles in Brownian motion when thelight hits the moving particles, thereby changing the wavelength(typically red light at 633 nm or near-infrared at 830 nm) of theincoming light—this change is related to the size of the particles. Theparticles in a liquid move about randomly and their motion is used todetermine the size of the particles: small particles are moving quicklyresulting in a more rapid intensity fluctuation, whereas large particlesare moving slowly, leading to slower intensity fluctuations.Construction of the time-dependent autocorrelation function from themeasured intensity fluctuation and fitting of this correlation curve toan exponential function gives a description of the particle motion inthe medium by calculation of the Diffusion coefficient of the Brownianmolecular motion. The hydrodynamic diameter of the particles cansubsequently be calculated by using the Stokes-Einstein equation. Forpolydisperse samples, this curve is a sum of exponential decays. Thepolydispersity index (PDI) is a parameter derived from the cumulantanalysis of the DLS measured intensity autocorrelation functionoriginally introduced by D. E. Koppel in The Journal of Chemical Physics57(11); 1972; pp: 4814-20. In the cumulant analysis, a singleexponential fit is applied to the resulting autocorrelation function bythe applied DLS software assuming a single-sized population following aGaussian distribution. The polydispersity index is related to thestandard deviation (σ) of the hypothetical Gaussian distribution aroundthe assumed particle size population in the following fashion:

PDI=σ² /Z _(D) ²,

where Z_(D) is Z-average size or cumulants mean, the intensity weightedmean hydrodynamic size of the ensemble collection of particle,representing the average of several species in the case of polydispersesamples (Stepto, RFT et al. (2009). “Dispersity in Polymer Science” PureAppl. Chem. 81 (2): 351-353).

Calculated polydispersity indices are dimensionless parametersrepresenting the width of the particle size distribution in thesolution. PDI values between 0.1 to 0.2 correspond to a narrow particlesize distribution approximately representing a monodisperse particlesize distribution. PDI values around 0.3 suggest an increasing width ofthe particle size distribution containing an increasing number ofdifferent particle populations. Values ranging between 0.5 and 0.7represent a very broad particle size distribution containing very largeparticles or aggregates. PDI values greater than 0.7 indicate the samplehas a very broad particle size distribution and may contain largeparticles or aggregates. In other words, the lower the PDI value, themore predominant infective viral particles species are present, i.e.viral particles species with a narrow particle size and without or withonly a small amount of aggregates and, accordingly, a higher efficacy ofthe viral vector composition can be achieved.

In accordance with the present invention, the PDI is less than 0.5. Asdescribed above, this PDI indicates a particle size distribution rangingfrom almost monodisperse to moderate polydisperse, with infectiveparticles as the predominant species and only a minor portion of largeparticles or agglomerates, or even without any large particles oragglomerates. Preferably, the PDI is less than 0.3, more preferably lessthan 0.2 and most preferably less than 0.1.

In accordance with the present invention and the applied example 5, thepreferred PDI value is less than 0.5 for enveloped viruses, e.g. MVA,and less than 0.3 for non-enveloped viruses, e.g. adenoviruses.Furthermore, it is preferred to maintain the above mentioned PDI valuesduring viral vector processing, manufacturing, and distribution phases.

The method of the present invention comprises in a first step (a) theprovision of replication-deficient viral vectors.

Replication-deficient viral vectors are viral vectors that are notcapable of replicating to generate new viral particles in host cells.For example, the viral vectors can have lost their replicationcompetence by empirical and rational attenuation processes resulting ina loss of important parts of their genome accompanied by (i) retentionof their ability to infect several cell types, and (ii) retention oftheir immunogenicity. Also VLPs fall under the term“replication-deficient viral vector”, in accordance with the presentinvention.

Due to the lack of replication competence, replication-deficient viralvectors represent safe and robust mechanism to induce both effector cellmediated and humoral immunity. As a consequence, priming with thesevectors can improve the magnitude, quality and durability of suchresponses, while at the same time providing an increased safety.

Suitable replication-deficient viral vectors for vaccine preparation arewell known in the art. For example, Verheust C. et al. (Vaccine 30,2012) provides a review regarding modified vaccinia Ankara virus(MVA)-based vectors, Rosewell A et al., (J Genet Syndr Gene Ther, 2011)provides a review regarding helper-dependent adenoviral vectors, andMulder AM et al. (PlosOne 7, 2012) provides a review regardingrecombinant VLP-based vaccines. The considerations for choosing asuitable viral vector for vaccine production commonly applied in the artapply mutatis mutandis with regard to choosing a suitable viral vectorfor vaccine production in accordance with the present invention.Accordingly, viral vectors already available in the art, as well asnovel viral vectors, may be employed in the claimed method. Preferably,the replication-deficient viral vectors are selected from the groupconsisting of MVA, adenovirus, adeno associated virus, lentivirus,Vesicular stomatitis virus, herpes simplex virus, or measles virus. Mostpreferably, the replication-deficient viral vector is modified vacciniaAnkara virus (MVA) or adenovirus.

The replication-deficient viral vectors can be freshly prepared, e.g.reconstituted after harvesting from cell cultures, or can be provided asa pre-prepared composition, for example from commercial sources.

In a second step (b), the method comprises the provision of a solutioncomprising at least one sugar and at least three different excipientsselected from hydrophilic and amphiphilic excipients, wherein theexcipients are characterized by polar, aliphatic, aromatic, negativelycharged, and/or positively charged functional groups.

The solution, in accordance with the present invention, can be anaqueous or a non-aqueous solution. In the context of the presentinvention, the term “aqueous solution” refers on one hand to water butextends on the other hand also to buffered solutions and hydrophilicsolvents miscible with water, thus being able to form a uniform phase.Examples for aqueous solutions include, without being limited, water,methanol, ethanol or higher alcohols as well as mixtures thereof.Non-limiting examples for non-aqueous solvents include dimethylsulfoxide(DMSO), ethylbenzene, and other polar solvents.

The term “comprising”, as used in accordance with the present invention,denotes that further steps and/or components can be included in additionto the specifically recited steps and/or components. However, this termalso encompasses that the claimed subject-matter consists of exactly therecited steps and/or components.

Non-limiting examples of further components that can be comprised in thesolution according to step (b) of the method of the invention includee.g., water, amino acids, buffers such as phosphate, citrate, succinate,acetic acid, histidine, glycine, arginine and other organic acids ortheir salts; antioxidants such as ascorbic acid, methionine, tryptophan,cysteine, glutathione, chelating agents such asethylenediaminetetraacetic acid (EDTA); counterions such as sodium;and/or nonionic surfactants such as polysorbates, poloxamers, or PEG orother solvents. Preferably, the solution does not contain any proteinsother than the (viral) proteins that are part of the viral vectors andthe above included components in form of a pharmaceutical carrier.

The solution according to step (b) of the method of the inventionfurther comprises at least one sugar.

The term “sugar”, as used herein, refers to any types of sugars, i.e.the monosaccharide, disaccharide or oligosaccharide forms ofcarbohydrates as well as sugar alcohols. Examples of suitable sugarsinclude, without being limiting, trehalose, saccharose, sucrose,glucose, lactose, mannitol, and sorbitol or sugar derivatives such asaminosugars, e.g glucosamine or n-acetyl glucosamine.

The term “at least”, as used herein, refers to the specifically recitedamount or number but also to more than the specifically recited amountor number. For example, the term “at least one” encompasses also atleast 2, at least 3, at least 4, at least 5, at least 6, at least 7, atleast 8, at least 9, at least 10, such as at least 20, at least 30, atleast 40, at least 50 and so on. Furthermore, this term also encompassesexactly 1, exactly 2, exactly 3, exactly 4, exactly 5, exactly 6,exactly 7, exactly 8, exactly 9, exactly 10, exactly 20, exactly 30,exactly 40, exactly 50 and so on.

It will further be appreciated that the term “one sugar” means one typeof sugar and does not limit the number of molecules of this particulartype of sugar to one. Further, in those cases where more than one sugaris comprised, such as e.g. two sugars, two different types of sugarenvisaged. Preferably, the solution comprises exactly one type of sugar,preferably trehalose.

Preferred amounts of sugars to be comprised in the solution according tothe invention are between 0.1 mg/ml to 200 mg/ml sugar, more preferablybetween 10 mg/ml to 180 mg/ml sugar, even more preferably between 20mg/ml to 160 mg/ml sugar and most preferably the amount is about 80mg/ml sugar. Where a mixture of different types of sugars is employed,these preferred amounts refer to the sum of all sugars in the solution.

The term “about”, as used herein, encompasses the explicitly recitedvalues as well as small deviations therefrom. In other words, an amountof sugar of “about 80 mg/ml” includes, but does not have to be exactlythe recited amount of 80 mg/ml but may differ by several mg/ml, thusincluding for example 92 mg/ml, 84 mg/ml, 88 mg/ml, 76 mg/ml, 72 mg/mlor 68 mg/ml. The skilled person is aware that such values are relativevalues that do not require a complete accuracy as long as the valuesapproximately correspond to the recited values. Accordingly, a deviationfrom the recited value of for example 15%, more preferably of 10%, andmost preferably of 5% is encompassed by the term “about”. Thesedeviations of 15%, more preferably of 10% and most preferably of 5% holdtrue for all embodiments pertaining to this invention wherein the term“about” is used.

Preferably, the amount of sugar is exactly 80 mg/ml.

In accordance with the present invention, the solution according to step(b) of the method of the invention further comprises at least threedifferent excipients selected from hydrophilic and amphiphilicexcipients, wherein the excipients are characterized by polar,aliphatic, aromatic, negatively charged, and/or positively chargedfunctional groups.

Excipients are well known in the art. Excipients are defined asingredients that are included in a composition, such as e.g.pharmaceutical compositions, together with the active agent. They aretypically added to formulations for several reasons and, thus, someexcipients may have more than one effect or purpose for being part ofthe formulation. One of their main functions is that of a stabilizer.The main function of such stabilizers in pharmaceutical formulations isto protect the biologically active agent against the different types ofstresses that are applied to said biologically active agent, such ase.g. a protein or a viral vector, during isolation, purification, dryinge.g. by lyophilization, spray-drying, spray-freeze drying orfoam-drying, storage either in solution or after drying as well asreconstitution after drying. There are specific mechanisms ofstabilization of biologically active agents, which are specificallyrelated to the excipients in the formulation. Stabilization is forexample achieved by strengthening of the stabilizing forces, bydestabilization of the denatured state, or by direct binding ofexcipients to the biologically active agents. Frequently employedexcipients for use as stabilizers of biologically active agents include,without being limiting, sugars, polyols, amino acids, amines, salts,polymers and surfactants, each of which may exert different stabilizingeffects.

Non-limiting examples of excipients selected from hydrophilic andamphiphilic excipients, wherein said excipients are furthercharacterized by having polar, aliphatic, aromatic, negatively charged,and/or positively charged functional groups, classified according tointernational pharmacopoeias as save excipients for use in viral vectorbased compositions are represented in Table 1 below.

TABLE 1 Excipients classified according to international pharmacopoeiasas save excipients for use in viral vector based compositions.ALPHA.-TOCOPHEROL .ALPHA.-TOCOPHEROL, DL-1,2-DIMYRISTOYL-SN-GLYCERO-3-(PHOSPHO-S-(1-GLYCEROL))1,2-DIMYRISTOYL-SN-GLYCERO-3-PHOSPHOCHOLINE1,2-DISTEAROYL-SN-GLYCERO-3-(PHOSPHO-RAC-(1-GLYCEROL))1,2-DISTEAROYL-SN-GLYCERO-3-PHOSPHOCHOLINE ACETIC ACID ACETIC ACID,GLACIAL ACETIC ANHYDRIDE ACETONE SODIUM BISULFITE ACETYLATEDMONOGLYCERIDES ACETYLTRYPTOPHAN, DL- ACTIVATED CHARCOAL ADIPIC ACIDALANINE ALBUMIN AGGREGATED ALBUMIN COLLOIDAL ALBUMIN HUMAN ALCOHOLALCOHOL, DEHYDRATED ALCOHOL, DENATURED ALCOHOL, DILUTED AMMONIUM ACETATEAMMONIUM HYDROXIDE AMMONIUM SULFATE ANHYDROUS CITRIC ACID ANHYDROUSDEXTROSE ANHYDROUS LACTOSE ANHYDROUS TRISODIUM CITRATE ARGININE ASCORBICACID ASPARTIC ACID BENZALKONIUM CHLORIDE BENZENESULFONIC ACIDBENZETHONIUM CHLORIDE BENZOIC ACID BENZYL ALCOHOL BENZYL BENZOATE BENZYLCHLORIDE BIBAPCITIDE BORIC ACID BROCRINAT BUTYLATED HYDROXYANISOLEBUTYLATED HYDROXYTOLUENE BUTYLPARABEN CALCIUM CALCIUM CHLORIDE CALCIUMGLUCEPTATE CALCIUM HYDROXIDE CALCOBUTROL CALDIAMIDE SODIUM CALOXETATETRISODIUM CALTERIDOL CALCIUM CAPTISOL CARBON DIOXIDECARBOXYMETHYLCELLULOSE CARBOXYMETHYLCELLULOSE SODIUM, UNSPECIFIED FORMCASTOR OIL CELLULOSE, MICROCRYSTALLINE CHLOROBUTANOL CHLOROBUTANOLHEMIHYDRATE CHLOROBUTANOL, ANHYDROUS CHOLESTEROL CITRATE CITRIC ACIDCITRIC ACID MONOHYDRATE CITRIC ACID, HYDROUS CORN OIL COTTONSEED OILCREATINE CREATININE CRESOL CROSCARMELLOSE SODIUM CROSPOVIDONE CYSTEINECYSTEINE HYDROCHLORIDE DALFAMPRIDINE DEOXYCHOLIC ACID DEXTRAN DEXTRAN 40DEXTROSE DEXTROSE MONOHYDRATE DEXTROSE SOLUTION DIATRIZOIC ACIDDIETHANOLAMINE DIMETHICONE MEDICAL FLUID 360 DIMETHYL SULFOXIDEDIPALMITOYLPHOSPHATIDYLGLYCEROL, DL- DISODIUM HYDROGEN CITRATE DISODIUMSULFOSALICYLATE DISOFENIN DISTEAROYLPHOSPHATIDYLCHOLINE, DL- DOCUSATESODIUM EDETATE CALCIUM DISODIUM EDETATE DISODIUM EDETATE DISODIUMANHYDROUS EDETATE SODIUM EGG PHOSPHOLIPIDS ETHANOLAMINE HYDROCHLORIDEETHYL ACETATE ETHYLENEDIAMINE ETHYLENE-VINYL ACETATE COPOLYMERS EXAMETAZI ME FERRIC CHLORIDE FRUCTOSE GADOLINIUM OXIDE GAMMA CYCLODEXTRINGELATIN GENTISIC ACID GENTISIC ACID ETHANOLAM IDE GENTISIC ACIDETHANOLAMINE GLUCEPTATE SODIUM GLUCEPTATE SODIUM DIHYDRATEGLUCONOLACTONE GLUCURONIC ACID GLUTATHIONE GLYCERIN GLYCINE GLYCINEHYDROCHLORIDE GUANIDINE HYDROCHLORIDE HETASTARCH HEXYLRESORCINOLHISTIDINE HUMAN ALBUMIN MICROSPHERES HYALURONATE SODIUM HYDROCHLORICACID HYDROCHLORIC ACID, DILUTED HYDROXYETHYLPIPERAZINE ETHANE SULFONICACID HYDROXYPROPYL .BETA.-CYCLODEXTRIN IODINE IODOXAMIC ACID IOFETAMINEHYDROCHLORIDE ISOLEUCINE ISOPROPYL ALCOHOL ISOTONIC SODIUM CHLORIDESOLUTION LACTIC ACID, DL- LACTIC ACID, L- LACTIC ACID, UNSPECIFIED FORMLACTOBIONIC ACID LACTOSE MONOHYDRATE LACTOSE, HYDROUS LACTOSE,UNSPECIFIED FORM LECITHIN LECITHIN, EGG LECITHIN, HYDROGENATED SOYLEUCINE LIDOFENIN LYSINE LYSINE ACETATE MAGNESIUM CHLORIDE MAGNESIUMSTEARATE MALEIC ACID MANNITOL MEBROFENIN MEDRONATE DISODIUM MEDRONICACID MEGLUMINE METACRESOL METAPHOSPHORIC ACID METHANESULFONIC ACIDMETHIONINE METHYL PYRROLIDONE METHYLBORONIC ACID METHYLCELLULOSESMETHYLENE BLUE METHYLPARABEN MIRIPIRIUM CHLORIDE MONOTHIOGLYCEROLN-(CARBAMOYL-METHOXY PEG-40)-1,2- DISTEAROYL-CEPHALIN SODIUMN,N-DIMETHYLACETAMIDE NIACINAMIDE NIOXIME NITRIC ACID NITROGEN OCTANOICACID OXIDRONATE DISODIUM OXYQUINOLINE PALMITIC ACID PEANUT OIL PEGVEGETABLE OIL PEG-20 SORBITAN ISOSTEARATE PEG-40 CASTOR OIL PEG-60CASTOR OIL PEG-60 HYDROGENATED CASTOR OIL PENTASODIUM PENTETATEPENTETATE CALCIUM TRISODIUM PENTETIC ACID PERFLUTREN PHENOL PHENOL,LIQUEFIED PHENYLALANINE PHENYLETHYL ALCOHOL PHENYLMERCURIC NITRATEPHOSPHATIDYL GLYCEROL, EGG PHOSPHOLIPID PHOSPHOLIPID, EGG PHOSPHORICACID POLOXAMER 188 POLYETHYLENE GLYCOL 200 POLYETHYLENE GLYCOL 300POLYETHYLENE GLYCOL 3350 POLYETHYLENE GLYCOL 400 POLYETHYLENE GLYCOL4000 POLYETHYLENE GLYCOL 600 POLYGLACTIN POLYLACTIDE POLYOXYETHYLENEFATTY ACID ESTERS POLYOXYL 35 CASTOR OIL POLYPROPYLENE GLYCOLPOLYSILO)KANE POLYSORBATE 20 POLYSORBATE 40 POLYSORBATE 80 POLYVINYLALCOHOL POTASSIUM BISULFITE POTASSIUM CHLORIDE POTASSIUM HYDROXIDEPOTASSIUM METABISULFITE POTASSIUM PHOSPHATE, DIBASIC POTASSIUMPHOSPHATE, MONOBASIC POVIDONE K12 POVIDONE K17 POVIDONES PROLINE PROPYLGALLATE PROPYLENE GLYCOL PROPYLPARABEN PROTAMINE SULFATE SACCHARINSODIUM SACCHARIN SODIUM ANHYDROUS SALT SERINE SESAME OIL SILICONESIMETHICONE SODIUM ACETATE SODIUM ACETATE ANHYDROUS SODIUM ASCORBATESODIUM BENZOATE SODIUM BICARBONATE SODIUM BISULFATE SODIUM BISULFITESODIUM CARBONATE SODIUM CARBONATE DECAHYDRATE SODIUM CARBONATEMONOHYDRATE SODIUM CHLORATE SODIUM CHLORIDE SODIUM CHLORIDE INJECTIONSODIUM CHLORIDE INJECTION, BACTERIOSTATIC SODIUM CHOLESTERYL SULFATESODIUM CITRATE SODIUM DESOXYCHOLATE SODIUM DITHIONITE SODIUMFORMALDEHYDE SULFOXYLATE SODIUM GLUCONATE SODIUM HYDROXIDE SODIUMHYPOCHLORITE SODIUM IODIDE SODIUM LACTATE SODIUM LACTATE, L- SODIUMMETABISULFITE SODIUM OLEATE SODIUM PHOSPHATE SODIUM PHOSPHATE DIHYDRATESODIUM PHOSPHATE, DIBASIC SODIUM PHOSPHATE, DIBASIC, ANHYDROUS SODIUMPHOSPHATE, DIBASIC, DIHYDRATE SODIUM PHOSPHATE, DIBASIC, DODECAHYDRATESODIUM PHOSPHATE, DIBASIC, HEPTAHYDRATE SODIUM PHOSPHATE, MONOBASICSODIUM PHOSPHATE, MONOBASIC, ANHYDROUS SODIUM PHOSPHATE, MONOBASIC,DIHYDRATE SODIUM PHOSPHATE, MONOBASIC, MONOHYDRATE SODIUM PHOSPHITESODIUM PYROPHOSPHATE SODIUM SUCCINATE HEXAHYDRATE SODIUM SULFATE SODIUMSULFATE ANHYDROUS SODIUM SULFITE SODIUM TARTRATE SODIUM THIOGLYCOLATESODIUM THIOMALATE SODIUM THIOSULFATE SODIUM THIOSULFATE ANHYDROUS SODIUMTRIMETAPHOSPHATE SORBITAN MONOPALMITATE SORBITOL SORBITOL SOLUTIONSOYBEAN OIL STANNOUS CHLORIDE STANNOUS CHLORIDE ANHYDROUS STANNOUSFLUORIDE STANNOUS TARTRATE STARCH STEARIC ACID STERILE WATER FORINHALATION STERILE WATER FOR INJECTION SUCCIMER SUCCINIC ACID SUCROSESULFOBUTYLETHER .BETA.-CYCLODEXTRIN SULFUR DIOXIDE SULFURIC ACIDSULFUROUS ACID TARTARIC ACID TARTARIC ACID, DL- TERT-BUTYL ALCOHOLTETRAKIS(2-METHOXYISOBUTYLISOCYANIDE) COPPER(I)TETRAFLUOROBORATETETROFOSMIN THEOPHYLLINE THIMEROSAL THREONINE TIN TRIFLUOROACETIC ACIDTRISODIUM CITRATE DIHYDRATE TROMANTADINE TROMETHAMINE TRYPTOPHANTYROSINE UREA URETHANE VALINE VERSETAMIDE YELLOW WAX ZINC ZINC ACETATEZINC CARBONATE ZINC CHLORIDE ZINC OXIDE

Preferred amounts of the sum of excipients to be comprised in thesolution according to the invention are between 0.001 and 100 mg/ml,preferably between 1 and 80 mg/ml, more preferably between 5 and 60mg/ml, even more preferably between 10 and 30 mg/ml and most preferablythe amount is about 20 mg/ml.

Preferably, the solution comprises trehalose or sucrose as the sugar andmannitol as the sugar alcohol and amino acids as the at least threeexcipients. Even more preferably, the solution comprises trehalose asthe sugar and at least three different amino acids as the at least threeexcipients.

Furthermore, the solution is characterized by an excipient to sugarratio of at least 1:2. More preferably, the solution is characterized byan excipient to sugar ratio of at least 1:1.5 (w/w), such as e.g. atleast 1:1(w/w) and most preferably of at least 1:0.1 (w/w).

Preferably, the pH value of the resulting composition according to step(b) will be adjusted to pH values between 4.0 and 9.0 before mixing withthe replication deficient viral vectors of step (a). The pH value chosendepends on the requirements for the particular viral vector, determinedby biologic characteristics such as e.g. size, enveloped (lipidmembrane) or not enveloped etc.

In a third step (c), the method of the present invention comprises thestep of mixing the replication deficient viral vectors of step (a) withthe solution of step (b).

The term “mixing”, as used herein, is not particularly limited andincludes all means of mixing viral vectors with a solution according to(b). For example, the components of step (a) and (b) can simply betransferred into the same vessel, where they can mix by diffusion; theycan additionally be stirred, e.g. by swirling the vessel around or bystirring with a suitable tool. Stirring can be for a limited amount oftime, such as e.g. once or twice, or can be continuously.

Preferably, the components of step (a) and (b) can mixed together byre-buffering of the composition of the recited step (a) in thecomposition of the recited step (b) using chromatographic operations aswell as dialysis, ultrafiltration and diafiltration operations.

The order of steps (a) and (b) is not particularly limited, i.e. step(a) can be carried out first, followed by step (b), or vice versa.Moreover, steps (a) and (b) can be carried out concomitantly. Step (c)is then carried out after steps (a) and (b) have been carried out.

In one embodiment, the method of the present invention consists of therecited steps (a) to (c). However, it will be appreciated that where themethod of the invention comprises (rather than consists of) the citedsteps (a) to (c), further method steps may be included in the method.For example, additional washing and/or drying steps may be included.Preferably, the method of the invention consists of the cited steps (a)to (c), optionally in combination with the below described additionalmethod steps (d) and (e), and optionally in combination with additionalwashing steps. Even more preferably, the method of the present inventionconsists of the recited steps (a) to (c), in combination with the belowdescribed additional method steps (d) drying and (e) reconstitution ofthe resulting dried composition.

In accordance with the present invention, a method is provided for thepreparation of improved viral vector-based vaccines and gene transfertherapeutics. By preparing vector-based compositions using the method ofthe present invention, unappreciated polydispersity can be avoided, thusresulting in a more suitable ratio between vector particle distributionand functional efficacy. Moreover, as discussed herein above, lowpolydispersity is associated with lower viscosity and not only providesbetter infectivity, but also leads to better syringeability andinjectability.

As shown in the appended Example 1, it was surprisingly observed thatmixing adenoviral vectors by diluting a highly concentrated adenoviralstock solution with a solution comprising at least one sugar and atleast three different excipients according to the invention, wherein thesolution is further characterized by an excipient to sugar ratio of atleast 1:2 (w/w) resulted in the complete retention of the hydrodynamicradii of the contained adenoviral particles monitored by Dynamic LightScattering (DLS) analysis compared to the similar dilutions in theoriginal supplier formulation and in the commonly usedphosphate-buffered saline (PBS).

Evaluation of the obtained correlation functions recorded in therespective DLS experiments for the adenoviral vector formulations beforefreeze drying suggested that already mixing of the adenoviral vectors inthe solutions according to the invention, particularly composition 1 andcomposition 2 led to a retention of the corresponding analyzedhydrodynamic radius of about 70 nm compared to the untreated stocksolution (Example 1; FIG. 1 A to C). In contrast, similar mixing of theadenoviral stock solution with PBS or with the original supplierformulation during the preparation process of the samples before freezedrying led to a remarkable increase in the measured hydrodynamic radiusof the adenoviral vectors compared to the untreated adenoviral vector(Example 1; FIG. 1A and FIG. 2).

In a preferred embodiment of the method of the invention, the at leastthree different excipients comprise amino acids. In an even morepreferred embodiment of the method of the invention, the at least threedifferent excipients are at least three different amino acids.

The term “amino acid”, as used herein, is well known in the art. Aminoacids are the essential building blocks of proteins. In accordance withthe present invention, the term “amino acid” refers to free amino acidswhich are not bound to each other to form oligo- or polymers such asdipeptides, tripeptides, oligopeptides or proteins (also referred toherein as polypeptides). The term “amino acid” includes naturallyoccurring amino acids, but also other amino acids such as artificialamino acids. They can be classified into the characteristic groups ofexcipients with non-polar, aliphatic; polar, uncharged; positivelyand/or negatively charged and/or aromatic R groups (Nelson D. L. & CoxM. M., “Lehninger Biochemie” (2005), pp. 122-127). The amino acidscomprised in the solution (b) of the present invention can be selectedfrom naturally occurring amino acids as well as artificial amino acidsor derivatives of these naturally occurring or artificial amino acids.

Naturally occurring amino acids include the 20 amino acids that make upproteins (i.e. the so-called proteinogenic amino acids), i.e. glycine,proline, arginine, alanine, asparagine, aspartic acid, glutamic acid,glutamine, cysteine, phenylalanine, lysine, leucine, isoleucine,histidine, methionine, serine, valine, tyrosine, threonine andtryptophan. Other naturally occurring amino acids are e. g. carnitine,creatine, creatinine, guanidinoacetic acid, ornithine, hydroxyproline,homocysteine, citrulline, hydroxylysine or beta-alanine. Artificialamino acids are amino acids that have a different side chain lengthand/or side chain structure and/or have the amine group at a sitedifferent from the alpha-C-atom. Derivates of amino acids include,without being limiting, n-acetyl-tryptophan, phosphonoserine,phosphonothreonine, phosphonotyrosine, melanin, argininosuccinic acidand salts thereof and DOPA. In connection with the present invention,all these terms also include the salts of the respective amino acids.

In an embodiment of the method of the invention, the at least threedifferent excipients comprise “at least one dipeptide and/ortripeptide”. Where more than one di- or tripeptide is comprised in thesolution, a mixture of dipeptides and tripeptides is explicitlyenvisaged herein. The number of di- and tripeptides can be selectedindependently of each other, e.g. the solution may comprise twodipeptides and three tripeptides. It will be readily understood by theskilled person that when referring to a certain number of di- andtripeptides herein, said number is intended to limit the amount ofdifferent types of di- and tripeptides, but not the number of moleculesof one type of dipeptide or tripeptide. Thus, for example the term “fourdipeptides or tripeptides”, refers to four different types of dipeptidesand/or tripeptides, wherein the amount of each individual di- and/ortripeptide is not particularly limited. Preferably, the number of(different) di- or tripeptides does not exceed nine di- or tripeptides.

The term “dipeptide or tripeptide”, as used herein, relates to peptidesconsisting of two or three amino acids, respectively. Exemplarydipeptides are glycylglutamine (Gly-Gln), glycyltyrosine (Gly-Tyr),alanylglutamine (Ala-Gln) and glycylglycine (Gly-Gly). Furthernon-limiting examples of naturally occurring dipeptides are carnosine(beta-alanyl-L-histidine), N-acetyl-carnosine(N-acetyl-(beta-alanyl-L-histidine), anserine (beta-alanyl-N-methylhistidine), homoanserine (N-(4-aminobutyryl)-L-histidine), kyotorphin(L-tyrosyl-L-arginine), balenine (or ophidine) (beta-alanyl-N tau-methylhistidine), glorin (N-propionyl-γ-L-glutamyl-L-ornithine-δ-lac ethylester) and barettin (cyclo-[(6-bromo-8-en-tryptophan)-arginine]).Examples of artificial dipeptides include, without being limiting,aspartame (N-L-a-aspartyl-L-phenylalanine 1-methyl ester) andpseudoproline.

Exemplary tripeptides are glutathione (γ-glutamyl-cysteinyl-glycine) andits analogues ophthalmic acid (L-γ-glutamyl-L-α-aminobutyryl-glycine) aswell as norophthalmic acid (y-glutamyl-alanyl-glycine). Furthernon-limiting examples of tripeptides include isoleucine-proline-proline(IPP), glypromate (Gly-Pro-Glu), thyrotropin-releasing hormone (TRH,thyroliberin or protirelin: L-pyroglutamyl-L-histidinyl-L-prolinamide),melanostatin (prolyl-leucyl-glycinamide), leupeptin(N-acetyl-L-leucyl-L-leucyl-L-argininal) and eisenin (pGlu-Gln-Ala-OH).

It is also envisaged herein that the solution of (b) comprises at leastthree excipients including (an) amino acid(s) as well as at least onedi- and/or tripeptide.

Preferably, the total amount of all amino acids, dipeptides and/ortripeptides (that is the sum of all of these components in the solution)to be employed is between 0.001 and 100 mg/ml, preferably between 1 and80 mg/ml, more preferably between 5 and 60 mg/ml, even more preferablybetween 10 and 30 mg/ml and most preferably the amount is about 20mg/ml.

It is preferred that the amino acids, and/or the di- and/or tripeptides,when used in connection with medical applications, do not exert anypharmacological properties.

In another preferred embodiment of the method of the invention, theviral vector-based composition is prepared for storage as a liquid. Inanother preferred embodiment of the method of the invention, the viralvector-based composition is prepared for storage as a dried preparation.Such viral vector-based composition (in liquid or dried preparations)may be subsequently used for the preparation of vaccines or genetransfer therapeutics.

In a further preferred embodiment of the method of the invention, whichembodiment comprises a further step (d) of drying the compositionobtained in step (c), the composition is dried by freeze-drying,spray-drying, spray freeze drying, or supercritical drying.

The term “drying”, as used herein, refers to the reduction or removal ofthe liquid content present in the composition. The liquid content isconsidered to have been reduced if the liquid is reduced to less than20%, such as for example less than 10%, such as for example less than8%, more preferably less than 7%, such as less than 5% or less than 1%.Even more preferably, the liquid is reduced to 0.5% or less.

Suitable methods for drying include, without being limiting,lyophilisation (freeze drying), spray drying, freeze-spray drying,convection drying, conduction drying, gas stream drying, drum drying,vacuum drying, dielectric drying (by e.g. radiofrequency or microwaves),surface drying, air drying or foam drying.

Freeze-drying, also referred to as lyophilisation, is also well known inthe art and includes the steps of freezing the sample and subsequentlyreducing the surrounding pressure while adding sufficient heat to allowthe frozen water in the material to sublime directly from the solidphase to the gas phase followed by a secondary drying phase. Preferably,the lyophilised preparation is then sealed to prevent the re-absorptionof moisture.

Spray-drying is also well known in the art and is a method to convert asolution, suspension or emulsion into a solid powder in one singleprocess step. Generally, a concentrate of the liquid product is pumpedto an atomising device, where it is broken into small droplets. Thesedroplets are exposed to a stream of hot air and lose their moisture veryrapidly while still suspended in the drying air. The dry powder isseparated from the moist air in cyclones by centrifugal action, i.e. thedense powder particles are forced toward the cyclone walls while thelighter, moist air is directed away through the exhaust pipes.

Spray-drying is often the method of choice, as it avoids the freezingstep and requires lower energy costs as compared to lyophilisation.Spray-drying has also been shown to be a particularly advantageousdrying procedure that is suitable for biomolecules, due to the shortcontact time with high temperature and its special process control.Thus, because spray-drying results in a dispersible dry powder in justone step it is often favoured to freeze drying when it comes to dryingtechniques for biomolecules.

Spray-freeze-drying is also well known in the art and is a method thatcombines processing steps common to freeze-drying and spray-drying. Thesample provided is nebulised into a cryogenic medium (such as e.g.liquid nitrogen), which generates a dispersion of shock-frozen droplets.This dispersion is then dried in a freeze dryer.

Supercritical drying is another technique well known in the art. Thismethod relies on high-temperature and high-pressure above the criticaltemperature (Tc) and critical pressure (pc) to change a liquid into agas wherein no phase boundaries are crossed but the liquid to gastransition instead passes through the supercritical region, where thedistinction between gas and liquid ceases to apply. The densities of theliquid phase and vapour phase become equal at the critical point ofdrying.

Preferably, the step (d) of drying the composition obtained in (c) is bylyophilisation.

As further shown in Example 1 below, it was surprisingly found thatcombining the adenoviral vectors by the method of the invention with therecited at least three excipients and sugar at a ratio of at least 1:2provides superior stability for the dried adenoviral vectorformulations. In Example 1, the combination of adenoviral vectorpreparation with the compositions, particularly composition 1 and 2according to the present invention already during early phasedownscaling steps and subsequent freeze drying resulted in the completeretention of the infective titer (Example 1; FIG. 3) and thehydrodynamic radii of the viral particles (Example 1; FIG. 4 A and B).In contrast, freeze drying of the corresponding adenoviral vectorpreparations in the original supplier formulation resulted insignificant loss of infectivity (Example 1; FIG. 3) and in increasedparticle size Example 1; FIG. 4 C). The similar sample preparationprocedure in combination with the common phosphate-buffered saline (PBS)resulted in complete loss of infectivity (Example 1; FIG. 3) and inmassive increase in particle size (Example 1 FIG. 4 D) and the formationof significant amounts of higher order aggregates, already after freezedrying. Most importantly, such dried formulations of biopharmaceuticaldrug substances or drug products are suitable for a variety of furtherhandling steps, such as aliquoting, distribution, shipment, storage etc.

In accordance with this preferred embodiment of the method of thepresent invention, a dry composition is obtained. It is particularlypreferred that the composition is a powder composition. In the case offreeze drying or spray-freeze drying, the resulting dried composition isautomatically obtained in the form of a powder. In those cases where thedry composition is not obtained as a powder, but instead in the form ofe.g. a dried cake, the skilled person is aware of how to further modifythe composition in order to obtain a powder.

Such an additional drying step can be advantageous, as intermolecularinteractions in a composition can lead e.g. to modified electrostaticinteractions of viral vectors resulting in a loss of particle integrityand function. The reduced water content within the composition inaccordance with this preferred embodiment reduces the molecular mobilitywithin the product, and thus helps to maintain particle integrity andfunction.

In another preferred embodiment of the method of the invention, themethod further comprises the step of subsequently storing the viralvector-based composition at a temperature selected from about −90° C. toabout 50° C. More preferably, the viral vector-based composition issubsequently stored at a temperature range selected from the groupconsisting of about −90° C. to about −70° C., about −30° C. to about−10° C., about 1° C. to about 10° C., about 15° C. to about 25° C. andabout 30° C. to about 50° C. Even more preferably, the viralvector-based composition is subsequently stored at a temperature rangeselected from the group consisting of about −85° C. to about −75° C.,about −25° C., to about −15° C., about 2° C. to about 8° C. and about20° C. to about 45° C. Most preferably, the viral vector-basedcomposition is subsequently stored at a temperature selected from about−80° C., about −20° C., room temperature, about 4° C., about 25° C. andabout 40° C.

As additionally shown in Example 1 below, it was surprisingly observed,that the differences between the in vitro infectivities of theadenoviral vectors freeze-dried in the compositions according to theinvention (composition 1 and 2) and the original supplier formulationand PBS, respectively already observed directly after freeze drying wereeven more striking after storage of the freeze-dried preparations atelevated temperatures. A complete loss of function of the viral vectorsfreeze-dried in the original supplier formulation was observed, similarto the results obtained in PBS (Example 1; FIG. 5 A and B). In contrast,even after storage at 25° C. or even at 40° C., the freeze-driedadenoviral vector compositions that were formulated in the stabilizingcompositions 1 and 2 early during the production process retained almostthe same viral activity as the positive control, i.e. the adenoviralvector prior to being freeze-dried (depicted as dashed line in thediagram of FIG. 5.

These results of the in vitro infectivity experiments correspond wellwith the DLS experiments performed in parallel. As examples, theevaluation of the recorded DLS correlation function after storage of thedried adenoviral vector compositions either in the composition 1 and 2according to the invention or in the original supplier formulation andPBS, respectively after storage for 14 days at 40° C. were depicted inFIG. 6. The storage of the dried adenoviral vector preparations instabilizing composition 1 and 2 led to retention of the determinedhydrodynamic radii of the adenoviral particles (Example 1; FIG. 6 A andB) in contrast to the stored adenoviral particles in the originalsupplier formulation and in PBS (Example 1; FIGS. 7 A and 7 B).

In a further preferred embodiment of the method of the invention, themethod further comprises a step (e) of reconstituting the compositionobtained after drying.

Reconstituting of the composition can be carried out by any means knownin the art, such as e.g. dissolving the dried composition in a suitablesolution. Non-limiting examples of suitable solutions include thesolution of step (b) used for mixing with the viral vector as well asany other solution known to be suitable for viral vector-basedcompositions, such as e.g. water for injection, buffered solutions,solutions comprising amino acids, sugars, buffers, surfactants ormixtures thereof.

In a further preferred embodiment of the method of the invention, theviral vector is selected from the group consisting of MVA, adenovirus,Adenovirus-associated virus (AAV), lentivirus, vesicular stomatitisvirus (VSV), or herpesviruses.

The Modified Vaccinia Ankara (MVA) virus is a highly attenuated strainof vaccinia virus that was developed towards the end of the campaign forthe eradication of smallpox in the seventies of the previous century.MVA was derived from Vaccinia strain Ankara by over 570 passages inchicken embryo fibroblast cells (CEF). This resulted in six majordeletions corresponding to the loss of about 10% of the vaccinia genome.The complete genomic sequence is known and has a length of 178 kpcorresponding to 177 genes. The numerous mutations explain theattenuated phenotype of MVA and its inability to replicate in mammaliancells. MVA is widely considered as the Vaccinia virus strain of choicefor clinical investigation because of its high safety profile. MVA hasbeen administered to numerous animal species including monkeys, mice,swine, sheep, cattle, horses, and elephants, with no local or systemicadverse effects. Over 120,000 humans have been safely and successfullyvaccinated against smallpox with MVA by intradermal, subcutaneous, orintramuscular injections. Studies in mice and nonhuman primates havefurther demonstrated the safety of MVA under conditions of immunesuppression. Compared to replicating vaccinia viruses, MVA providessimilar or higher levels of recombinant gene expression even innon-permissive cells. In animal models, recombinant MVA vaccines havebeen found immunogenic and to protect against various infectious agentsincluding influenza, parainfluenza, measles virus, flaviviruses, andplasmodium parasites. The combination of a very good safety profile andthe ability to deliver antigens in a highly immunogenic way makes MVAsuitable as a vaccine vector.

Adenoviruses are medium-sized (90-100 nm), nonenveloped (naked)icosahedral viruses composed of a nucleocapsid and a double-strandedlinear DNA genome. There are over 51 different serotypes in humans,which are responsible for 5-10% of upper respiratory infections inchildren, and many infections in adults as well. When these virusesinfect a host cell, they introduce their DNA molecule into the host. Thegenetic material of the adenoviruses is not incorporated (transient)into the host cell's genetic material. The DNA molecule is left free inthe nucleus of the host cell, and the instructions in this extra DNAmolecule are transcribed just like any other gene. The only differenceis that these extra genes are not replicated when the cell is about toundergo cell division so the descendants of that cell will not have theextra gene. As a result, treatment with the adenovirus will requirere-administration in a growing cell population although the absence ofintegration into the host cell's genome should prevent the type ofcancer seen in the SCID trials. This vector system has shown realpromise in treating cancer and indeed the first gene therapy product(Gendicine) to be licensed is an adenovirus to treat cancer.

Viruses of the family adenoviridae infect various species of animals,including humans. Adenoviruses represent the largest non-envelopedviruses because they are the maximum size able to be transported throughthe endosome (i.e. envelope fusion is not necessary). The virion alsohas a unique “spike” or fiber associated with each penton base of thecapsid that aids in attachment to the host cell via thecoxsackie-adenovirus receptor on the surface of the host cell.

Adeno-associated virus (AAV) is a small virus that infects humans andsome other primate species. AAV is not currently known to cause diseaseand, consequently, the virus causes a very mild immune response. AAV caninfect both dividing and non-dividing cells and can incorporate itsgenome into that of the host cell. Moreover, episomal AAV elicits longand stable expression and, thus, AAV is suitable for creating viralvectors for gene therapy. Because of its potential use as a gene therapyvector, AAV has previously been modified (self-complementaryadeno-associated virus; scAAV). Whereas AAV packages a single strand ofDNA and requires the process of second-strand synthesis, scAAV packagesboth strands which anneal together to form double stranded DNA. Thisapproach allows for rapid expression in the target cell.

Lentiviruses, a subclass of retroviruses have recently been adapted asviral vectors for gene delivery because of their unique ability tointegrate into the genome of non-dividing cells. The viral genome in theform of RNA is reverse-transcribed when the virus enters the cell toproduce DNA, which is then inserted into the genome by the viralintegrase enzyme. The vector, now called a provirus, remains in thegenome and is passed on to the progeny of the cell when it divides.

Vesicular stomatitis Indiana virus (VSIV) (often still referred to asVSV) is a virus in the family Rhabdoviridae; the well-known rabies virusbelongs to the same family. VSIV can infect insects, cattle, horses andpigs. It has particular importance to farmers in certain regions of theworld where it can infect cattle and lead to diseases similar to thefoot and mouth disease virus.

Herpes viruses belong to the Herpesviridae, a large family of DNAviruses that cause diseases in animals and humans. Herpes simplexviruses (HSV) HSV-1 and HSV-2 (orolabial herpes and genital herpes),Varicella zoster virus (VZV; chicken-pox and shingles), Epstein-Barrvirus (EBV; mononucleosis), and Cytomegalovirus (CMV) are widespreadamong humans. More than 90% of adults have been infected with at leastone of these, and a latent form of the virus remains in most people.Herpes viruses are currently used as gene transfer vectors due to theirhigh transgenic capacity of the virus particle allowing to carry longsequences of foreign DNA, the genetic complexity of the virus genome,allowing to generate many different types of attenuated vectorspossessing oncolytic activity, and the ability of HSV vectors to invadeand establish lifelong non-toxic latent infections in neurons fromsensory ganglia from where transgenes can be strongly and long-termexpressed. Three different classes of vectors can be derived from HSV:replication-competent attenuated vectors, replication-incompetentrecombinant vectors and defective helper-dependent vectors known asamplicons. Replication-defective HSV vectors are made by the deletion ofone or more immediate-early genes, e.g. ICP4, which is then provided intrans by a complementing cell line. Oncolytic HSV vectors are promisingtherapeutic agents for cancer. Such HSV based vectors have been testedin glioma, melanoma and ovarian cancer patients.

It is particularly preferred that the viral vector is MVA.

The above listed preferred viral vectors have been evaluated regardingtheir safety profile in animals and/or humans and preclinical andclinical data are available, respectively.

In another preferred embodiment of the method of the invention, thereplication-deficient viral vector is a virus like particle.

Virus like particles (VLPs) provide the advantage that they are notinfectious and do not contain viral genetic material. Accordingly, theyare not associated with any risk of reassembly as is possible when liveattenuated viruses are used as viral vectors.

In another preferred embodiment of the method of the invention, themethod further comprises adding an antigenic polypeptide.

An “antigenic polypeptide” in accordance with the present invention isnot particularly limited, as long as it elicits an immune response. Theantigenic polypeptide can be selected from e.g. viruses, bacteria, ortumor cells. For example, the antigenic polypeptide can be a viralsurface protein of a virus other than the viral vector employed in themethod of the invention, or a part thereof; or a main immunogenic viralprotein or part thereof. These additional antigenic polypeptides can forexample be used for priming the immune system in a prime-boostvaccination. In that case, the boost reaction is elicited by therespective viral vector or VLP relied on for preparing the viralvector-based composition by the method of the present invention. Theterm “polypeptide” as used herein interchangeably with the term“protein” describes linear molecular chains of amino acids, includingsingle chain proteins or their fragments.

The step of adding the antigenic polypeptide can be carried out atdifferent time points. For example, the antigenic polypeptide can beadded to the replication-deficient viral vector provided in step (a).Alternatively, the antigenic polypeptide can be additionally admixed instep (c) or be added to the resulting composition subsequently to themixing in step (c). Furthermore, as an additional alternative, theantigenic polypeptide can be added to the viral vector-based compositionafter reconstitution in step (e).

In a further preferred embodiment of the method of the invention, themethod further comprises adding at least one adjuvant.

Adjuvants as well as their mode of action are well known in the art.Some adjuvants, such as alum and emulsions (e.g. MF59®), function asdelivery systems by generating depots that trap the antigenic substanceat the injection site, providing slow release in order to provide acontinued stimulation of the immune system. These adjuvants enhance theantigen persistence at the injection site and increase the recruitmentand activation of antigen presenting cells (APCs). Particulate adjuvants(e.g. alum) have the capability to bind antigenic substances to formmulti-molecular aggregates which will encourage uptake by APCs. Someadjuvants are also capable of directing antigen presentation by themajor histocompatibility complexes (MHC). Other adjuvants, essentiallyligands for pattern recognition receptors (PRR), act by inducing theinnate immunity, predominantly targeting the APCs and consequentlyinfluencing the adaptive immune response. Members of nearly all of thePRR families are potential targets for adjuvants. These includeToll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-I-likereceptors (RLRs) and C-type lectin receptors (CLRs). They signal throughpathways that involve distinct adaptor molecules leading to theactivation of different transcription factors. These transcriptionfactors (NF-KB, IRF3) induce the production of cytokines and chemokinesand IL-18.

Preferably, the at least one adjuvant is selected from Alum, MF59®,AS03, AF03, AS04, RC-529, Virosomen, ISCOMATRIX®, CpG 1018, CpG 7909,Vaxlmmune, ProMune®, IC-31®, CTA1-DD or Cyclic di-AMP. These adjuvants,their class, indications and provider as well as product names aresummarized in Table 2 below.

TABLE 2 Detailed informations on particularly preferred adjuvants.Adjuvant Class Main indications Provider/Product Alum Aluminium saltsvarious various Aluminiumhydroxid world-wide AluminiumphosphateAluminiumhydroxyphosphate MF59 ^(®) Oil-in-Water emulsion SeasonalNovartis/Fluad 4.3% Squalen Influenza 0.5% Polysorbat 80 0.5%Sorbitantriolate (Span 85 ^(®)) 10 mM sodiumcitrate AS03 Oil-in-Wateremulsion Pandemic GSK/Pandemrix 10.69 mg Squalene Influenza 11.86 mgD,L-α-Tocopherol (Vit. E) 4.86 mg Polysorbate 80 AF03 Oil-in-Wateremulsion Pandemic Sanofi Pasteur/ 12.4 mg Squalene Influenza Humanza 1.9mg Sorbitanoleate 2.4 mg Polysorbate 20 2.3 mg Mannitol AS04 KombinationHepatitis B virus GSK/Fendrix Monophposphoryllipid A Human Cervarix undAluminiumsalz Papillomavirus RC-529 Combination synthetic Hepatitis Bvirus Dynavax monophposphoryllipid A and aluminiumsalt VirosomenPhosphatidylcholine bilayer Hepatitis A virus Crucell/ liposomes 150 nmSeasonal Inflexal V Influenza ISCOMATRIX ^(®) ISCOM various CSL LimitedImmunostimulating Parkville, Complex Victoria, Antigen AustralianCholesterol Phospholipid Saponin from Quillaja Saponaria CpG 1018Oligodeoxynukleotide Hepatitis B virus Dynavax/ HEPLISAV-B Cancer SD-101CpG 7909 Oligodeoxynukleotide Cancer Coley/ VaxImmune vaccinationChiron/Pfizer ProMune ^(®) Hepatitis B virus GSK Treatment of CancerIC-31 ^(®) Peptide and Tuberkulosis Intercell OligodesoxynukleotidCTA1-DD Fusion protein from CTA1- MIVAC Domaine of Cholera ToxinsDevelopment (CT) with maintaining ADP- AB in Sweden ribosylatingenzymatic function and a dimer from Ig binding domain of Protein A(S.aureus) as target domaine

The step of adding the adjuvant can be carried out at different timepoints. For example, the adjuvant can be added to thereplication-deficient viral vector provided in step (a). Alternatively,or additionally, the adjuvant can be admixed in step (c) or it can beadded to the resulting composition subsequently to the mixing in step(c). As a further alternative or additional option, it can be added tothe viral vector-based composition after reconstitution in step (e).

In another preferred embodiment of the method of the invention, at leastone of the adjuvants is a saponine. Alternatively, the adjuvant is amixture of substances comprising a saponine.

Saponines are a class of chemical compounds forming secondarymetabolites which are found in natural sources, derived from naturalsources or can be chemically synthesised. Saponines are found inparticular abundance in various plant species. Saponines are amphipathicglycosides grouped phenomenologically by the soap-like foaming theyproduce when shaken in aqueous solutions, and structurally by theircomposition of one or more hydrophilic glycoside moieties combined witha lipophilic steroidal or triterpenoid aglycone. Their structuraldiversity is reflected in their physicochemical and biologicalproperties. Non-limiting examples of saponines are glycyrrhizic acid,glycyrrhetinic acid, glucuronic acid, escin, hederacoside and digitonin.

Preferably, the saponine is selected from well-known adjuvantcompositions, e.g., the saponine extracted from Quillaja Saponaria, aslisted in Table 2, without being limiting.

In another embodiment, the saponine is glycyrrhizic acid or a derivativethereof. Glycyrrhizic acid is also known as glycyrrhicic acid,glycyrrhizin or glycyrrhizinic acid. Glycyrrhizic acid is water-solubleand exists as an anion that can be a potential ligand to formelectrostatically associated complexes with cationic molecules of activeingredients. Without wishing to be bound by theory, the presentinventors hypothesise that the anionic glycyrrhizic acid forms complexeswith amino acids present in the solution of the present invention (i.e.arginine, or lysine) through electrostatic interactions, hydrogen bondsor both.

Derivatives of glycyrrhizic acid are well-known in the art and includethose produced by transformation of glycyrrhizic acid on carboxyl andhydroxyl groups, by conjugation of amino acid residues into thecarbohydrate part or the introduction of2-acetamido-β-d-glucopyranosylamine into the glycoside chain ofglycyrrhizic acid. Other derivatives are amides of glycyrrhizic acid,conjugates of glycyrrhizic acid with two amino acid residues and a free30-COOH function and conjugates of at least one residue of amino acidalkyl esters in the carbohydrate part of the glycyrrhizic acid molecule.Examples of specific derivatives can be found e. g. in Kondratenko etal. (Russian Journal of Bioorganic Chemistry, Vol 30(2), (2004), pp.148-153).

Preferred amounts of glycyrrhizic acid (or derivatives thereof) to beemployed are between 0.01 and 15 mg/ml, preferably between 0.1 and 10mg/ml, more preferably between 0.5 and 5 mg/ml, even more preferablybetween 1 and 3 mg/ml and most preferably the amount is 2 mg/ml.

As is known in the art, saponines, in particular glycyrrhizic acid, havebeen found to be advantageously present in function of an adjuvant, asthey enhance the immunogenic effect of the viral vector basedcomposition.

In another preferred embodiment of the method of the invention, thereplication-deficient viral vectors of (a) are replication-deficientviral vectors that have been reconstituted immediately after harvestingfrom cell cultures and purification.

Means and methods for reconstituting replication-deficient viral vectorsare well known in the art. For example, after amplification of areplication-deficient viral vector, such as e.g. MVA, in the appropriatecell culture model, crude stock preparations of MVA can be semi-purifiedfrom cell debris and recombinant proteins by ultracentrifugation througha sucrose cushion. After discarding the supernatant (cell debris andsucrose) the pelleted viral vector material can be mixed with a solutionaccording to (b). Alternatively, to obtain more highly purified viruses,the semi-purified material can be centrifuged through a 25-40% sucrosegradient. The viral vector band appearing at the lower half of the tubeis concentrated and the remaining sucrose is simultaneously removed byfilling an ultracentrifuge tube with the solution according to (b),pelleting the viral vector material by ultracentrifugation andsuspending the pellet in a solution according to (b).

The decrease in the amount of infectious particles present in acomposition as compared to non-infectious particles due to an increasingpolydispersity starts immediately after harvesting viral particles fromcell culture. Thus, it is particularly preferred in accordance with thepresent invention that the replication-deficient viral vectors are mixedwith the solution of (b) as early as possible after the initialharvesting of the viral vectors.

As shown in e.g. example 2 below, the early addition of the stabilizingcompositions, particularly composition 1 and 2 can have a strong impacton the stability of the adenoviral vector preparation during its entirepreparation procedure. The in vitro infectivity analysis of theadenoviral preparations after re-buffering by dialysis in composition 1and 2, respectively either directly after the purification step by CsCIdensity ultracentrifugation (process step 1), or later in thepreparation process (process step 2) revealed differences between thetwo applied stabilizing compositions. Re-buffering in composition 1according to the preparation in process step 1 and 2 led to the completeretention of the infectivity directly after dialysis (Example 2; FIG. 9)and after application of 5 and 10 freeze and thaw cycles compared to thepositive control depicted as dashed line (Example 2; FIG. 11 A and B).Re-buffering in composition 2 during an earlier step of the preparationprocess (process step 1) led also to complete retention of theinfectivity directly after dialysis and minor loss of the infectivetiter after application of repeated freeze and thaw cycles (Example 2;FIG. 9 and FIG. 11 A). In contrast, re-buffering in composition 2 duringpreparation in process step 2 led to a remarkable reduction in theinfective titer already directly after the dialysis Example 2; FIG. 9).Further application of repeated freeze and thaw cycles resulted in afurther, significant decrease of the infective titer (Example 2; FIG. 11B).

Thus, the early application of the stabilizing composition according tothe invention was found to have a pronounced stabilizing effect on theparticular biomolecule during the entire production process.

These results of the in vitro infectivity experiments correspond wellwith the DLS experiments performed in parallel. In FIG. 10 A and B theresults of the determination of the hydrodynamic radii of the adenoviralparticles after the preparation in composition 1 and 2 during processstep 1 indicate the complete retention of the adenoviral particle sizeafter preparation of the formulations directly after the purificationstep using ultracentrifugation which is in line with the infectivityresults in FIG. 9. In the case of composition 1, after re-buffering theadenoviral vector preparation according to process step 2 a slightincrease of the hydrodynamic particle radii was observed (FIG. 10 C)which is in accordance with the infectivity results shown in FIG. 9. Incontrast, re-buffering of the adenoviral vector preparation incomposition 2 corresponding to processing step 2 resulted in aremarkable increase of the hydrodynamic radius of the adenoviralparticles (FIG. 10 D) accompanied by the formation of higher orderaggregates that may explain the loss of function in the in vitroinfectivity tests (FIG. 9).

After five and ten repeated freeze and thaw cycles, changes in thehydrodynamic radii of viral particles, particularly in composition 2were measured by DLS. No remarkable increase was observed in composition1 after five and even after ten freeze and thaw cycles when preparedduring process steps 1 and 2 (Example 2; FIGS. 12 A and B and 13 A andB). When composition 2 was used during process step 1 the hydrodynamicradii already after five freeze and thaw cycles were remarkablyincreased in conjunction with the formation of higher order aggregates(Example 2; 13 C and D) and were not measurable after ten freeze andthaw cycles and when used in process step 2 due to further increasedradii and higher order aggregates which were outside the DSL measurelimit.

The present invention further relates to a viral vector-basedcomposition obtained or obtainable by the method of the invention.

These viral vector-based compositions may be used for anti-bacterial,antiviral, anti-cancer, anti-allergy vaccination and/or for genetransfer therapy for the treatment of diseases with a geneticbackground.

In a preferred embodiment, the composition is a pharmaceuticalcomposition.

In accordance with the present invention, the term “pharmaceuticalcomposition” relates to a composition for administration to a patient,preferably a human patient.

The pharmaceutical compositions can be administered to the subject at asuitable dose. The dosage regimen will be determined by the attendingphysician and clinical factors. As is well known in the medical arts,dosages for any one patient depend upon many factors, including thepatient's size, body surface area, age, the particular compound to beadministered, sex, time and route of administration, general health, andother drugs being administered concurrently. The therapeuticallyeffective amount for a given situation will readily be determined byroutine experimentation and is within the skills and judgment of theordinary clinician or physician. The pharmaceutical composition may befor administration once or for a regular administration over a prolongedperiod of time. Generally, the administration of the pharmaceuticalcomposition should be in the range of for example 1 μg/kg of body weightto 50 mg/kg of body weight for a single dose. However, a more preferreddosage might be in the range of 10 μg/kg to 20 mg/kg of body weight,even more preferably 100 μg/kg to 10 mg/kg of body weight and even morepreferably 500 μg/kg to 5 mg/kg of body weight for a single dose.

The components of the pharmaceutical composition to be used fortherapeutic administration must be sterile. Sterility is readilyaccomplished for example by filtration through sterile filtrationmembranes (e.g., 0.2 pm membranes).

The various components of the composition may be packaged as a kit withinstructions for use.

Accordingly, the present invention further relates to a kit comprising aviral vector-based composition obtained or obtainable by the method ofthe invention and, optionally, instructions how to use the kit.

Whereas the term “kit” in its broadest sense does not require thepresence of any other compounds, vials, containers and the like otherthan the recited components, the term “comprising”, in the context ofthe kit of the invention, denotes that further components can be presentin the kit. Non-limiting examples of such further components includeantigenic polypeptides or adjuvants, as defined above, as well aspreservatives, buffers for storage, enzymes etc.

Where several components are comprised in the kit, the variouscomponents of the kit may be packaged in one or more containers such asone or more vials. Consequently, the various components of the kit maybe present in isolation or combination. The containers or vials may, inaddition to the components, comprise preservatives or buffers forstorage. In addition, the kit can contain instructions for use.

In a preferred embodiment of the kit of the invention, the kit comprisesa viral vector-based composition obtained or obtainable by the method ofthe invention and, in the same or a separate container, an antigenicpolypeptide. These separate containers with i) the viral vector-basedcomposition and ii) the antigenic polypeptide can be used in separatevaccination steps (either simultaneously or subsequently to each other),e.g. for a prime-boost immunization approach.

In an alternative or additional preferred embodiment of the kit of theinvention, the kit comprises a viral vector-based composition obtainedor obtainable by the method of the invention and, in the same or aseparate container, one or more adjuvants.

Also envisaged is a kit, comprising (i) a viral vector-based compositionobtained or obtainable by the method of the invention; (ii) an antigenicpolypeptide and (iii) one or more adjuvants, in the same or differentcontainers.

The present invention also relates to the viral vector-based compositionof the invention for use as a prime-boost vaccine.

The “prime-boost vaccine strategy” is well known in the art andencompasses a first step of “priming” an immune response, followed by asecond step of “boosting” the previously primed immune response. Thisapproach enables high levels of antigen specific T-cell memory as wellas protective cellular immunity to pathogens, even in humans, and thusis a promising approach in vaccination (Woodland D L, Trends inImmunology, 2004; Nolz J C, Harty J T. Adv Exp Med Biol. 2011;780:69-83. doi: 10.1007/978-1-4419-5632-3_7. Strategies and implicationsfor prime-boost vaccination to generate memory CD8 T cells).

Viral vector-based compositions are highly attractive for therapeuticprime-boost vaccine approaches. For example, prophylactic vaccinationfor the prevention of HBV infection is well established. In contrast, aneffective therapy of chronic hepatitis due to HBV infection and itssequelae is currently not available and might be successfully addressedby a prime-boost vaccination strategy with a specific antigen prime anda subsequent specific viral vector-based boost that induces antigenspecific antibody production as well as antigen specific T cellresponses both resulting in a highly efficient vaccination outcome. Asdiscussed herein above, the data provided in the appended Examples showthat the biological, immunogenic activity of a viral vector-basedcomposition prepared by the method of the present invention is improvedas compared to viral vector-based compositions prepared by othermethods. In other words, the ability of the inventive viral vector-basedcompositions to stimulate the immune system of a subject, such as e.g.to elicit cytotoxic T lymphocytes (CTL) of the immune system to protectthe subject against the disease for which the vaccine has beendeveloped, is improved.

In a further preferred embodiment of the invention, the viralvector-based composition is for intramuscular, subcutaneous,intradermal, transdermal, oral, peroral, nasal, and/or inhalativeapplication.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In case of conflict, the patentspecification, including definitions, will prevail.

Regarding the embodiments characterized in this specification, inparticular in the claims, it is intended that each embodiment mentionedin a dependent claim is combined with each embodiment of each claim(independent or dependent) said dependent claim depends from. Forexample, in case of an independent claim 1 reciting 3 alternatives A, Band C, a dependent claim 2 reciting 3 alternatives D, E and F and aclaim 3 depending from claims 1 and 2 and reciting 3 alternatives G, Hand I, it is to be understood that the specification unambiguouslydiscloses embodiments corresponding to combinations A, D, G; A, D, H; A,D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B,D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C,D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C,F, I, unless specifically mentioned otherwise.

Similarly, and also in those cases where independent and/or dependentclaims do not recite alternatives, it is understood that if dependentclaims refer back to a plurality of preceding claims, any combination ofsubject-matter covered thereby is considered to be explicitly disclosed.For example, in case of an independent claim 1, a dependent claim 2referring back to claim 1, and a dependent claim 3 referring back toboth claims 2 and 1, it follows that the combination of thesubject-matter of claims 3 and 1 is clearly and unambiguously disclosedas is the combination of the subject-matter of claims 3, 2 and 1. Incase a further dependent claim 4 is present which refers to any one ofclaims 1 to 3, it follows that the combination of the subject-matter ofclaims 4 and 1, of claims 4, 2 and 1, of claims 4, 3 and 1, as well asof claims 4, 3, 2 and 1 is clearly and unambiguously disclosed.

The above considerations apply mutatis mutandis to all appended claims.To give a non-limiting example, the combination of claims 12, 8, 4, 3and 1 is clearly and unambiguously envisaged in view of the claimstructure. The same applies for example to the combination of claims 12,8 and 1 etc.

The invention is illustrated with the following figures which show:

FIG. 1: Dynamic Light Scattering (DLS) determination of the hydrodynamicradii of the adenoviral vector compositions before freeze drying as amodel for aggregation and polydispersity in a suspension. (A) Evaluationof the correlation function recorded in the DLS experiments using aregularization fit by the DynaPro DLS software of the adenoviral stocksolution as a control, (B) evaluation of the correlation function of theadenoviral vector preparation directly after mixing with composition 1by dilution and (C) evaluation of the correlation function of theadenoviral vector preparation directly after mixing with composition 2by dilution. The calculated hydrodynamic radii of the adenoviral vectorpreparations in composition 1 and 2 are in line with the measured radiiof the adenoviral particles in the untreated stock solution and withvalues known from the literature.

FIG. 2: Dynamic Light Scattering (DLS) determination of the hydrodynamicradii of the adenoviral vector compositions before freeze drying as amodel for aggregation and polydispersity in a suspension. (A) Evaluationof the correlation function recorded in the DLS experiments using aregularization fit by the DynaPro DLS software of the adenoviral vectorpreparation directly after mixing with the original supplier formulationand (B) evaluation of the correlation function of the adenoviral vectorpreparation directly after mixing with PBS. In contrast to the previousfigure, the hydrodynamic radii of the adenoviral particles after mixingwith the original supplier formulation and with PBS are increasedcompared to the untreated stock solution.

FIG. 3: In vitro infectivity of adenoviral vectors after freeze dryingin different formulations as a model for functionality under freezedrying stress conditions. Adenoviral vector preparations were formulatedby dilution and subsequently freeze-dried in composition 1 and 2. Afterreconstitution of the freeze-dried vectors an in vitro infectivity assayin HEK 293 cells was carried out using an antibody based colorimetricdetection of the adenoviral Hexon protein to indicate a successfulamplification of the adenovirus in the infected cells. A completeretention of the infective titers of the adenoviral vector preparationsformulated in composition 1 and 2 was observed (infective units per mlas compared to positive control; depicted as dashed line). In contrast,freeze drying of the adenoviral vectors diluted in the original supplierformulation led to a remarkable loss of the infective titers and freezedrying of the adenoviral vectors diluted in PBS resulted in a completeloss of the corresponding infective titers.

FIG. 4: Dynamic Light Scattering (DLS) determination of the hydrodynamicradii of the adenoviral particles in the corresponding adenoviral vectorpreparations after freeze drying as a model for aggregation andpolydispersity under freeze drying stress conditions. (A) Evaluation ofthe correlation function recorded in the DLS experiment using aregularization fit by the DynaPro DLS software of the adenoviral vectorpreparation after freeze drying in composition 1, (B) evaluation of theadenoviral vector preparation after freeze drying in composition 2. Thecalculated hydrodynamic radii of the adenoviral vector preparations incomposition 1 and 2 are in line with the measured radii of theadenoviral particles in the untreated stock solution (FIG. 1 A) and withvalues known from the literature.

FIG. 5: Dynamic Light Scattering (DLS) determination of the hydrodynamicradii of the adenoviral particles in the corresponding adenoviral vectorpreparations after freeze drying as a model for aggregation andpolydispersity under freeze drying stress conditions. (A) Evaluation ofthe correlation function recorded in the DLS experiment using aregularization fit by the DynaPro DLS software of the adenoviral vectorpreparation directly after freeze drying in the original supplierformulation and (B) evaluation of the correlation function of theadenoviral vector preparation directly after freeze drying in PBS. Incontrast to the previous figure, the hydrodynamic radii of theadenoviral particles after freeze drying in the original supplierformulation and in PBS are increased associated with the formation ofhigher order aggregates compared to the untreated stock solution (FIG. 1A).

FIG. 6: In vitro infectivity of adenoviral vectors after freeze dryingin different formulations and subsequent storage of the driedformulations at elevated temperatures as a model for functionality underthermal stress conditions. t=0 d (black bars on the left) shows the invitro infectivity directly after freeze drying and reconstitution beforestorage. The dashed line shows the corresponding infective titer of theuntreated positive control. (A) In vitro infectivity of the adenoviralvector compositions after re-buffering by dilution in composition 1 and2 and subsequent storage of the freeze-dried formulations for 21 days(set of bars in the middle) and 42 days (set of bars on the right) at25° C. and at 60% residual humidity, as compared to the originalsupplier buffer and PBS. (B) In vitro infectivity of the adenoviralvector compositions after re-buffering by dilution in composition 1 and2 and subsequent storage of the freeze-dried formulations for 7 days(set of bars in the middle) and 28 days (set of bars on the right) at40° C. and at 75% residual humidity, as compared to the originalsupplier buffer and PBS. Complete retention of the adenoviralinfectivity was observed in the samples prepared in the compositions 1and 2, whereas storage in either the original supplier buffer or in PBSled to the complete loss of adenoviral infectivity.

FIG. 7: Dynamic Light Scattering (DLS) determination of the hydrodynamicradii of the adenoviral particles in the corresponding adenoviral vectorpreparations after freeze drying and subsequent storage for 14 days at40° C. as a model for aggregation and polydispersity under thermalstress conditions. (A) Evaluation of the correlation function recordedin the DLS experiment using a regularization fit by the DynaPro DLSsoftware of the adenoviral vector preparation after storage of the driedformulations for 14 days at 40° C. in composition 1 and (B) evaluationof the adenoviral vector preparation after storage of the driedformulations for 14 days at 40° C. in composition 2. The calculatedhydrodynamic radii of the adenoviral vector preparations in composition1 and 2 are in line with the measured radii of the adenoviral particlesin the untreated stock solution (FIG. 1 A) and with values known fromthe literature.

FIG. 8: Dynamic Light Scattering (DLS) Determination of the hydrodynamicradii of the adenoviral particles in the corresponding adenoviral vectorpreparations after freeze drying as a model for aggregation andpolydispersity under thermal stress conditions. (A) Evaluation of thecorrelation function recorded in the DLS experiment using aregularization fit by the DynaPro DLS software of the adenoviral vectorpreparation after freeze drying and subsequent storage for 14 days at40° C. in the original supplier formulation and (B) evaluation of thecorrelation function of the adenoviral vector preparation after freezedrying and subsequent storage for 14 days at 40° C. in PBS. In contrastto the previous figure, the hydrodynamic radii of the adenoviralparticles after freeze drying and subsequent storage at elevatedtemperature in the original supplier formulation and with PBS areincreased compared to the untreated stock solution associated with theformation of higher order aggregates.

FIG. 9: In vitro infectivity of adenoviral vector preparations afterformulation in stabilizing compositions 1 and 2 prepared during eitherprocess step 1 or process step 2 as a model for functionality underthermal stress conditions. Adenoviral preparations were re-buffered bydialysis in composition 1 and 2, respectively either directly after thepurification step by CsCI density ultracentrifugation (process step 1),or later in the preparation process (process step 2). In process step 1,a complete retention of the infective titer after dialysis in bothcompositions was observed compared to the positive control (depicted asdashed line). In contrast, dialysis during process step 2 led to aremarkable loss of infective titers of nearly two log levels whencarried out in composition 2, whereas dialysis in composition 1 led tothe complete retention of the infective titer, similar to the resultsobtained for process step 1.

FIG. 10: Dynamic Light Scattering (DLS) determination of thehydrodynamic radii of the adenoviral particles in the correspondingadenoviral vector preparations after formulation in stabilizingcompositions 1 and 2 during either process step 1 or process step 2, asa model for aggregation and polydispersity. Re-buffering of theadenoviral vector particle preparations in composition 1 using dialysiseither in process step 1 or 2 resulted in the retention of thehydrodynamic radii of the particles (A) and (C). Re-buffering of theadenoviral particles in composition 2 during preparation in process step1 led to the complete retention of the hydrodynamic radius of theadenoviral vector (B). In contrast, re-buffering of the adenoviralparticles in composition 2 during preparation in process step 2 led toan increase in the hydrodynamic radius of the particles and theassociated formation of large aggregates (D).

FIG. 11: In vitro infectivity of the adenoviral vector preparationsafter repeatedly applied freeze and thaw cycles as a model forfunctionality under stress conditions. (A) Re-buffering of theadenoviral vector preparations by dialysis during preparation in processstep 1. (B) Re-buffering of the adenoviral vector preparations bydialysis during preparation in process step 2. In both preparationprocedures (process step 1 and 2), re-buffering in composition 1 led tothe complete retention of the infectivity directly after dialysis(initial titer) and after application of 5 and 10 freeze and thaw cycles(A) and (B) compared to the positive control depicted as dashed line.Re-buffering in composition 2 during an earlier step of the preparationprocess (process step 1) led also to complete retention of theinfectivity directly after dialysis (initial titer; A, left set of bars)and minor loss of the infective titer after application of repeatedfreeze and thaw cycles (A). In contrast, re-buffering in composition 2during preparation in process step 2 led to a remarkable reduction inthe infective titer already directly after the dialysis (B; left set ofbars). Further application of repeated freeze and thaw cycles resultedin a further, significant decrease of the infective titer (B; middle andright set of bars).

FIG. 12: Dynamic Light Scattering (DLS) Determination of thehydrodynamic radii of the adenoviral particles in the stabilizingcompositions 1 after application of either five or ten freeze and thawcycles as a model for aggregation and polydispersity under stressconditions. Re-buffering of the adenoviral vector particle preparationsin composition 1 using dialysis in process step 2 resulted in theretention of the hydrodynamic radii of the particles (A) after theapplication of five freeze and thaw cycles and (B) after application often freeze and thaw cycles.

FIG. 13: Dynamic Light Scattering (DLS) Determination of thehydrodynamic radii of the adenoviral particles in stabilizingcompositions 1 and 2 during either process step 1 or process step 2after application of five freeze and thaw cycles as a model foraggregation and polydispersity under stress conditions. Re-buffering ofthe adenoviral vector particle preparations in composition 1 usingdialysis either in process step 1 or 2 resulted in the retention of thehydrodynamic radii of the particles after the application of five freezeand thaw cycles (A) and (B). In contrast, re-buffering of the adenoviralparticles in composition 2 either during preparation in process step 1or 2 led to an increase in the hydrodynamic radii of the particles andthe associated formation of higher order aggregates after theapplication of five freeze and thaw cycles (C) and (D).

FIG. 14: Transmission Electron Microscopy (TEM) of an adenoviral vectorpreparation after freeze drying and reconstitution in composition 1. (A)low magnification; (B) intermediate magnification; (C) highmagnification; (D) special observation with extra high magnification.Adenovirus particles appeared as icosahedral-shaped bright intactparticles (black arrow). A small number of more densely stained lesspronounced icosahedral-shape particles (white arrow) were observed,putatively representing partially destabilized virions. Small lightlystained structures (white arrowhead) are present in the background ofthe grid, but no significant presence of debris or Adenovirus subunitswere observed. Adenovirus particles preferentially appeared as singleicosahedral-shaped bright intact entities and no aggregation ofAdenovirus particles or debris was observed.

FIG. 15: Transmission Electron Microscopy (TEM) of an adenoviral vectorpreparation after freeze drying and reconstitution in composition 2. (A)low magnification; (B) intermediate magnification; (C) highmagnification; (D) special observation with extra high magnification.Adenovirus particles appeared as icosahedral-shaped bright intactparticles (black arrow). The background appeared very smooth with nosignificant presence of debris or Adenovirus subunits. Adenovirusparticles preferentially appeared as single icosahedral-shaped brightintact entities and no aggregation of Adenovirus particles or debris wasobserved.

FIG. 16: Transmission Electron Microscopy (TEM) of an adenoviral vectorpreparation after freeze drying and reconstitution in PBS. (A) lowmagnification; (B) intermediate magnification; (C) high magnification;(D) special observation with extra high magnification. No intactAdenovirus particles were observed. Small ring-like structures (blackarrowhead) possibly representing hexon structures were occasionallyobserved on the grid (black arrowhead). The hexon structures were bothobserved as free entities and bound to small clusters of debris (blackarrow or to spherical lightly stained structures (D)). A small number oflarger aggregates containing debris were observed (B).

FIG. 17: Transmission Electron Microscopy (TEM) of an adenoviral vectorpreparation after freeze drying and reconstitution in the originalsupplier formulation. (A) low magnification; (B) intermediatemagnification; (C) high magnification; (D) special observation withextra high magnification. No intact Adenovirus particles were observed.Small ring-like structures (black arrowhead) possibly representing hexonstructures were occasionally observed on the grid (black arrowhead). Thehexon structures were both observed as free entities and bound to smallclusters of debris (black arrow or to spherical lightly stainedstructures (D)).

FIG. 18: Transmission Electron Microscopy (TEM) of a adenoviral vectorpreparation stored at −80° C. in a standard buffer as a positivecontrol. (A) low magnification; (B) intermediate magnification; (C) highmagnification; (D) special observation overview image at lowmagnification. Adenovirus particles were observed, both asicosahedral-shaped bright intact particles (A; black arrow) and denselystained less pronounced icosahedral-shape particles (A, B and C; whitearrow), putatively representing partially disassembled particles. Thediameter of the Adenovirus particles was measured to approximately 100nm (vertex-to-vertex). The background shows the presence of debris (B;black dashed arrow), fiber structures (C; white dashed arrow) and smallring-like structures (C; black arrowhead), mainly as single entities andonly rarely in clusters, possibly representing hexon structures (C;inset). The Adenovirus particles appeared both as single entities and insmaller clusters. One larger aggregate containing Adenovirus and debriswas observed (D).

FIG. 19: In vitro infectivity of adenoviral vectors after liquid storageat 5° C., 25° C. and 37° C. in different formulations as a model forfunctionality under liquid stress conditions. Adenoviral vectorpreparations were formulated by dilution to 1×10⁸ IFU/ml in differentformulations and subsequently 100 μl were liquid stored in sterilePCR-tubes. After storage at different temperatures an in vitroinfectivity assay in HEK 293 cells was carried out using an antibodybased colorimetric detection of the adenoviral Hexon protein to indicatea successful amplification of the adenovirus in the infected cells. (A)After 3 months at 5° C. a complete retention of the infective titers ofthe adenoviral vector preparations formulated in composition 3 to 9 wasobserved (infective units per ml as compared to positive control;depicted as dashed line). In contrast, the original supplier formulation1 led to a higher loss of the infective titers of almost 2-log titers.(B) After 3 months liquid storage at 25° C. the infective titers of theadenoviral vector preparations formulated in composition 3 to 9 wasdecreased to 1-log titer, whereby the original supplier formulation 1led to a complete loss of the infective titers (infective units per mlas compared to positive control; depicted as dashed line). (C) After 35days at 37° C. the composition 8 is still detectable with a titer of1×10⁵ IFU/ml, whereby in the original supplier formulation 1 theadenoviral vector has no infective units already after 14 days at 37° C.(D) Dynamic Light Scattering (DLS) determination of the PDI values ofthe adenoviral particles in the corresponding adenoviral vectorpreparations after liquid storage at 37° C. as a model for increasingparticle size distributions expressed in PDI values under liquid storagestress conditions.

FIG. 20: Transmission Electron Microscopy (TEM) of an adenoviral vectorpreparation formulated in composition 8 after liquid storage for 28 daysat 25° C. (A) low magnification; (B) intermediate magnification; (C)high magnification; (D) special observation overview image at lowmagnification. Adenovirus particles were observed, both asicosahedral-shaped bright intact particles and densely stained lesspronounced icosahedral-shape particles and, putatively representingpartially disassembled particles. The diameter of the Adenovirusparticles was measured to approximately 100 nm (vertex-to-vertex). Thebackground shows the presence of debris, fiber structures and smallring-like structures, mainly as single entities and only rarely inclusters, possibly representing hexon structure).

FIG. 21: Transmission Electron Microscopy (TEM) of an adenoviral vectorpreparation formulated in composition 6 after liquid storage for 28 daysat 25° C. (A) low magnification; (B) intermediate magnification; (C)high magnification; (D) special observation overview image at lowmagnification. Adenovirus particles were observed, both asicosahedral-shaped bright intact particles and densely stained lesspronounced icosahedral-shape particles and, putatively representingpartially disassembled particles. The diameter of the Adenovirusparticles was measured to approximately 100 nm (vertex-to-vertex). Thebackground shows the presence of debris, fiber structures and smallring-like structures, mainly as single entities and only rarely inclusters, possibly representing hexon structure).

FIG. 22: In vitro infectivity of adenoviral vectors after liquid storageat 37° C. in composition 10 as a model for functionality under liquidstress conditions. The adenoviral vector preparation in composition 10showed a better retention of the infective titre after liquid storagefor 14 days and more pronounced for 21 days at 37° C. than thecorresponding preparation in the original supplier formulation 2.

FIG. 23: In vitro infectivity of adenoviral vectors after liquid storageat 37° C. in composition 11 and 12 or in original supplier formulations1 and 2 prepared by processing step 1 and 2 (PS1 and PS2) as a model forfunctionality during different processing steps. The adenoviral vectorpreparation in composition 11 and 12 (A) showed a better retention ofthe infective titre after liquid storage for 28 days at 37° C. than thecorresponding preparation in the original supplier formulations 1 and 2.The adenoviral vector preparations formulated in composition 11 usingpreparation step 2 showed a remarkable stabilization during storage for28 days at 37° C. compared to PBS (B; loss of infectivity already after14 days).

FIG. 24: In vitro infectivity of adenoviral vectors after application ofdifferent freeze-thaw cycles in compositions 11 and 12 after formulationaccording to PS1 and PS2 compared to the original supplier formulations1 and 2 and PBS. The adenoviral vector preparation in composition 11 and12 (A) showed a better retention of the infective titre afterapplication of 5 freeze-thaw cycles, and more pronounced after 15freeze-thaw cycles compared to the original supplier formulations 1 and2 and PBS. The adenoviral vector preparation formulated in composition11 using preparation step 2 (PS2) showed a better retention of theinfectivity after the application of 10 freeze-thaw cycles compared toPBS.

FIG. 25: Dynamic Light Scattering (DLS) determination of the PDI valuesof the adenoviral particles in the corresponding adenoviral vectorpreparations after the application of several freeze-thaw cycles as amodel for increasing particle size distributions expressed in PDI valuesunder different processing conditions. The PDI values of the adenoviralvector compositions formulated in compositions 11 and 12 according toPS1 were lower than 0.3 even after twenty freeze-thaw cycles (A).Formulation of the adenoviral vector samples in compositions 11 and 12according to PS2 resulted in retention of PDI values smaller than 0.3(B).

FIG. 26: Dynamic Light Scattering (DLS) determination of the PDI valuesof the MVA particles in composition 13. After application of 20freeze-thaw cycles, the PDI values of MVA in composition 13 were smallerthan 0.5 compared to original supplier formulations 1 and 2 and PBS.

The examples illustrate the invention:

EXAMPLE 1

The in vitro study of the functional and structural integrity offreeze-dried and subsequently stored adenoviral vectors showed that acomposition comprising amino acids and sugar stabilizes the viralvectors during freeze drying

1.1 Materials and Methods

Composition 1 and 2 contained the 7 amino acids alanine, arginine,glycine, glutamic acid, lysine, histidine and tryptophan in aconcentration corresponding to the sum of the amino acids of 40 g/I. Butin composition 1, a 5 fold increase of the tryptophan concentration anda 1.667 fold increase of the histidine and glutamic acid concentrationunder reduction of the concentrations of the other amino acids arginine,glycine, lysine and the retention of the alanine concentration comparedto composition 2 resulted in the same concentration according to the sumof amino acids of 40 g/l. Further, an additional surfactant polysorbate80 in a concentration of 0.05 g/l was added to composition 1 in contrastto composition 2. Both compositions contained trehalose as thecorresponding sugar in an amino acid to trehalose ratio of 1:2. The pHvalue was adjusted in all compositions to 7.

An adenoviral stock solution stored at −80° C. with a concentration of7.5*10¹⁰ IFU/ml in the original supplier formulation (Firma Sidon;Martinsried/Munich; Germany) was employed.

1.1.1 Sample Preparation and Freeze Drying

The adenoviral vector stock solution was re-buffered by dilution of thestock solution to a concentration of 1*10⁸ IFU/ml with eithercomposition 1 or composition 2. For comparison the stock solution wasdiluted with either the original supplier formulation or with PBS to thesame concentrations.

In order to prepare the samples for freeze drying, the differentadenoviral formulations were aliquoted in volumes of 500 μl in 2R freezedrying vials (Schott AG; Mainz; Germany) and subsequently freeze-driedusing the following drying parameters:

Protocol Step Target T (° C.) Slope (h) Hold (h) Pressure (mbar)Introduction 20 0 0 1000 Freezing −50 2:00 2:00 1000 Sublimation −500:01 0:30 0.045 −35 3:00 30:00 0.045 Secondary Drying 20 3:00 7:00 0.009

After freeze drying, the samples were visually inspected and one part ofthe samples was stored for a short time at 2-8° C. until analysis of theinitial infective titer at the time point t=0. The other part of thesamples was stored according to the guidelines of the InternationalCouncil for Harmonization (ICH) for 21 or 42 days at 25° C. underenvironmental conditions of 60% residual humidity, or for 7 or 28 daysat 40° C. under environmental conditions of 75% residual humidity.

1.1.2 Determination of the Infective Titers for Adenoviral Vectors inCell Culture

In order to analyze the infective titer of the adenoviral vectorformulations, an antibody based virus titration experiment in HEK 293cell culture using the detection of the adenoviral Hexon protein aftersuccessful amplification of the adenovirus in the infected cells wasapplied. 2.5*10⁵ HEK 293 (CCS) cells (Firma Sirion; Martinsried/Munich;Germany) were seeded per well of a 24-well micro titer plate in a volumeof 500 μl. The adenoviral vector formulations were reconstituted eitherdirectly after freeze drying or at the indicated time points uponstorage at 25° C. and at 40° C. As a positive control an aliquot of theadenoviral stock solution stored at −80° C. with a concentration of7.5*10¹⁰ IFU/ml in the original supplier formulation (Firma Sirion;Martinsried/Munich; Germany) was used. Subsequently, serial dilutions ofthe adenoviral samples were prepared and 50 μl of the resultingdilutions per well were used for infection of the cells. The plates wereincubated for 42 hours at 37° C. After infection, cells were fixed withmethanol, incubated with the primary anti-Hexon protein antibody (SantaCruz Biotechnology, Inc.; Dallas; Tex.: USA) , subsequently incubatedwith an horse radish peroxidase (HRP)-conjugated secondary anti-mouseantibody (Cell Signaling Technology; Danvers; Mass.; USA) specific forthe primary antibody and an HRP enzymatic reaction with diaminobenzidine(Carl Roth GmbH and Co.KG; Grafrath; Germany) was carried out, wherein abrown colouring indicates infected cells. The number of infected cellswas quantified by counting the brown coloured cells under themicroscope, wherein each infected cell is counted as one infectiousviral particle.

1.1.3 Dynamic Light Scattering (DLS) Measurement

DLS was carried out on samples taken before freeze drying directly afterre-buffering compared to an untreated positive control corresponding toan aliquot of the adenoviral stock solution stored at −80° C. as well ason samples after reconstitution of the adenoviral vector formulations.In the latter case, DLS was carried out either immediately after freezedrying (t=0) or at the relevant time points upon storage at 25° C. (21days, 42 days) and at 40° C. (7 days, 28 days).

To this end, 5 μl of the samples were pipetted into a special DLScuvette and analysed in a DynaPro Nanostar DLS instrument (WyattTechnology Europe GmbH; Dernbach; Germany). For each experimentalformulation, a blank measurement was performed under the sameconditions. The DLS measurements were performed with acquisition timesbetween 20 and 40 seconds in 10 or 20 cycles. The resulting correlationcurves were analysed using the DynaPro DLS software.

1.1.4 Transmissional Electron Microscopy

Adenoviral vector preparations were formulated by dilution andsubsequently freeze-dried in composition 1 and 2 as well as in originalsupplier formulation and PBS. The EM images were acquired by Vironova(Sweden). After reconstitution of the freeze-dried vectors 3 μl of thesample were applied onto a suitable hydrophilized EM grid (e.g.continuous carbon) washed with water, and negatively stained using 2%uranyl acetate. The grids were imaged using a FEI Tecnai G2 SpiritBiotwin electron microscope run at 100 kV accelerating voltage. Both lowand high magnification images were acquired in representative areas. Inthe case of the positive control 3 μl of the undiluted frozen storedsample (−80° C.) in the original supplier formulation buffer wereapplied onto the grid.

1.2 Results

Interestingly, the evaluation of the correlation functions recorded inthe DLS experiments directly after mixing of the adenoviral vectorpreparations with the solutions according to the invention (composition1 or composition 2) suggested a complete retention of the hydrodynamicradii of the adenoviral vectors (FIG. 1 B and C) as compared to those ofthe untreated adenoviral particles in the original stock solution (FIG.1 A). Similar mixing of the adenoviral stock solution by dilution withthe original supplier formulation or with PBS during the preparationprocess of the samples before freeze drying already led to a remarkableincrease in the measured hydrodynamic radii of the adenoviral vectors(FIG. 2 A and B) compared to the untreated adenoviral vector (FIG. 1 A).

The in vitro infectivity assay after freeze drying revealed that aformulation of adenoviral vector preparations in the stabilizingcompositions 1 and 2 early in the production process of a freeze-driedbiopharmaceutical product resulted in infective titers that correspondto those of the positive control depicted as dashed line in FIG. 1.Thus, a complete retention of infective titers was observed after freezedrying. In contrast, when the adenoviral vectors re-buffered in theoriginal supplier formulation were freeze-dried, a remarkable loss ofthe infective titers was observed and freeze drying in PBS even resultedin a complete loss of the corresponding infective titers (FIG. 3).

The in vitro infectivity results of the adenoviral preparations afterreconstitution of the dried products were well in line with the resultsof the parallel determination of the hydrodynamic radii by Dynamic LightScattering experiments. The combination of adenoviral vector preparationwith the composition 1 and 2 according to the present invention alreadyduring early phase downscaling steps and subsequent freeze dryingresulted in the complete retention of the hydrodynamic radii of theviral particles (Example 1; FIG. 4 A and B). In contrast, freeze dryingof the corresponding adenoviral vector preparations in the originalsupplier formulation resulted in increased particle sizes (Example 1;FIG. 5 A). The similar sample preparation procedure in combination withthe common phosphate-buffered saline (PBS) resulted in massive increasein particle size (Example 1 FIG. 5 B) and the formation of significantamounts of higher order aggregates, already after freeze drying.

These differences were even more striking after storage of thefreeze-dried preparations. A complete loss of function of the viralvectors freeze-dried in the original supplier formulation (FIG. 6) wasobserved, similar to the results obtained in PBS. In contrast, evenafter storage at 25° C. or even at 40° C., the freeze-dried adenoviralvector compositions that were formulated in the stabilizing compositions1 and 2 early during the production process retained almost the sameviral activity as the positive control, i.e. the adenoviral vector priorto being freeze-dried (depicted as dashed line in the diagram of FIG.6).

These results of the in vitro infectivity experiments correspond wellwith the DLS experiments performed in parallel. As examples, theevaluation of the recorded DLS correlation function after storage of thedried adenoviral vector compositions either in the composition 1 and 2according to the invention or in the original supplier formulation andPBS, respectively after storage for 14 days at 40° C. were depicted inFIGS. 7 and 8. The storage of the dried adenoviral vector preparationsin stabilizing composition 1 and 2 led to retention of the determinedhydrodynamic radii of the adenoviral particles (Example 1; FIGS. 7 A and7 B) in contrast to the stored adenoviral particles in the originalsupplier formulation and in PBS (Example 1; FIGS. 8 A and 8 B).

The freeze-dried adenovirus preparations were reconstituted and werefurther characterized using electron microscopic analysis. This analysisfurther substantiated that a combination of the adenoviral vectors withthe recited at least three excipients and sugar at a ratio of at least1:2, in accordance with the invention, provides superior stability forthe dried adenoviral vector formulations and also confirmed the abovedetailed infectivity and DLS results.

The electron microscopic images of the corresponding adenoviralpreparations in composition 1 and 2 (FIGS. 14 and 15) show relativelyevenly distributed Adenovirus particles, with the majority of theAdenovirus particles appearing as icosahedral-shaped bright intactparticles (black arrow) of approximately 100 nm diameter. A small numberof more densely stained, less pronounced icosahedral-shaped particleswas observed (white arrow), which presumably represent partiallydestabilized virions,. Small, lightly stained structures (whitearrowhead) are present in the background of the grid, but no significantpresence of debris or Adenovirus subunits was observed in composition 1(FIG. 14B and 14C). For composition 2, the background appeared verysmooth with no significant presence of debris or Adenovirus subunits(FIG. 15). In the Adenovirus preparations formulated in composition 1and 2, the Adenovirus particles preferentially appeared as singleicosahedral-shaped bright intact entities and no aggregation ofAdenovirus particles or debris was observed. In contrast, the analysisof the adenoviral vectors after freeze drying in the original supplierformulation (FIG. 17) and in PBS (FIG. 16) showed that no intactAdenovirus particles were observed. Small ring-like structures (blackarrowhead), possibly representing hexon structures, were occasionallyobserved on the grid (black arrowhead FIG. 16C and 17D), both as freeentities and bound to small clusters of debris (black arrow FIG. 16A and17A and B) or to spherical lightly stained structures (FIG. 16D and17D). In the corresponding adenoviral preparation in PBS a small numberof larger aggregates containing debris was observed (FIG. 16B).

For comparison, FIG. 18 shows the electron microscopic analysis of theremarkable higher concentrated positive control stored at −80° C. in astandard buffer. Adenovirus particles were observed, both asicosahedral-shaped bright intact particles (FIG. 18A; black arrow) anddensely stained less pronounced icosahedral-shape particles (FIG. 18A, Band C; white arrow), putatively representing partially disassembledparticles. The diameter of the Adenovirus particles was measured toapproximately 100 nm (vertex-to-vertex). The background shows thepresence of debris (FIG. 14B, black dashed arrow), fiber structures(FIG. 180; white dashed arrow) and small ring-like structures (FIG. 180;black arrowhead), mainly as single entities and only rarely in clusters,possibly representing hexon structures (FIG. 180; inset). The Adenovirusparticles appeared both as single entities and in smaller clusters. Onelarger aggregate containing Adenovirus and debris was observed (FIG.18D). It should be noted that the positive control was measured with theadenoviral composition in a standard buffer stored at −80° C. containingan infective titer of 2*10¹¹ IU/ml. In contrast, the infective titer ofthe freeze-dried and reconstituted adenoviral preparations according tothe EM images in FIGS. 14 to 17 was around 1*10⁸ IU/ml.

EXAMPLE 2

The in vitro study of the functional and structural integrity ofdifferent adenoviral vector preparations after freeze and thaw stressshowed that a composition comprising amino acids and sugar stabilizesthe viral vectors during freeze and thaw cycles

2.1 Materials and Methods 2.1.1 Sample Preparation and FurtherProcessing

High titers of adenoviral vector stocks of the adenoviral type 5 vectorscontaining the coding DNA for the eGFP protein 5*10⁸ HEK293 cells weretransduced with adenoviral particles. 48 h after transduction, the cellswere harvested and the release of viral particles was performed viaNa-Deoxycholat and DNase I treatment. Viral particles were purified byCsCI gradient ultracentrifugation usually followed by buffer exchange inthe original supplier formulation on PD10 columns and subsequentdetermination of the infective titer. The resulting high titeradenoviral stocks were subsequently aliquoted and stored at −80° C.

Sample preparation - process step 1: Adenoviral vector formulations wereprepared by re-buffering of the adenoviral vector preparationsimmediately after CsCI gradient ultracentrifugation. The obtainedadenoviral vector band was harvested and dialysed at 2-8° C. in eithercomposition 1 or 2 (as described in 1.1). The resulting formulationswere aliquoted and stored at −80° C.

Sample preparation—process step 2: Frozen (−80° C.) adenoviral stocksolutions (7.5*10¹⁰ IFU/ml; Sirion, Martinsried/Munich, Germany) werethawed (room temperature; RT) in the original supplier buffer andsubsequently dialysed at 2-8° C. in compositions 1 and 2.

2.1.2 Repeated Freeze and Thaw Cycles with Adenoviral Samples fromProcess Step 1 And Step 2 Preparations

In order to analyze the stability of the adenoviral vector preparationsduring subsequent stress conditions, 50 μl of the adenoviral vectors,formulated in composition 1 or 2 were subjected to repeated freeze (−80°C.) and thaw (RT) cycles. The in vitro infectivity (described in 1.1.2)was determined at the initial time point t=0 and after 5 and 10 freezethaw cycles by virus titration in HEK 293 cell cultures (described in1.1.2). In parallel, the hydrodynamic radii of the adenoviral particleswere measured by DLS (described in 1.1.3).

2.2 Results

The in vitro infectivity assay revealed that composition 1 fullyretained the infective titers of both adenoviral vector preparationsfrom process step 1 and step 2 (FIG. 9) compared to the positive control(dashed line in FIG. 9). Re-buffering of the adenoviral vectorpreparations immediately after the ultracentrifugation step (processstep 1) in composition 2 also fully retained the infectivity of theadenoviral vector preparation. Interestingly, composition 2 used afterprocess step 2, resulted in a loss of approximately two log levels ofthe initial titer (FIG. 9).

Upon additional freeze and thaw cycles (five and ten), composition 1retained the full infective titer, regardless of the production processstep and time point of re-buffering (FIG. 11 A and B). In contrast,composition 2 resulted in remarkably different effects when prepared inthe two different process steps 1 and 2. The infective titers ofcomposition 2 samples obtained according to process step 2 significantlyfurther decreased after five and even stronger after ten freeze and thawcycles (FIG. 11 B). When the adenoviral vectors were formulated at theearlier process step 1 in composition 2, only a minor titer loss wasobserved after five freeze and thaw cycles. Ten freeze and thaw cyclesresulted in a stronger decrease but to a minor extent compared to thepreparation in process step 2 (FIG. 11 A).

In parallel to the determination of the infective titers before andafter repeated freeze and thaw cycles, the hydrodynamic radii of thecorresponding adenoviral particles were analyzed using Dynamic LightScattering (DLS) (FIGS. 10, 12 and 13). Re-buffering of the adenoviralvector preparation directly after the purification step usingultracentrifugation (preparation step 1) resulted in the completeretention of the hydrodynamic radii of the viral particles in bothcompositions (FIGS. 10 A and 10 B) confirming the complete retention ofthe corresponding in vitro infectivity (FIG. 9). In the case ofcomposition 1, after re-buffering the adenoviral vector preparationaccording to process step 2 a slight increase of the hydrodynamicparticle radii was observed (FIG. 10 C) which is in accordance with theinfectivity results shown in FIG. 9. In contrast, re-buffering of theadenoviral vector preparation in composition 2 corresponding toprocessing step 2 resulted in a remarkable increase of the hydrodynamicradius of the adenoviral particles (FIG. 10 D) accompanied by theformation of higher order aggregates that may explain the loss offunction in the in vitro infectivity tests (FIG. 9).

After five and ten repeated freeze and thaw cycles, changes in thehydrodynamic radii of viral particles, particularly in composition 2were measured by DLS. No remarkable increase was observed in composition1 when prepared during process steps 1 and 2. As an example the DLSresults for the size of the adenoviral particles in composition 1 afterthe application of five and ten freeze and thaw cycles are depicted inFIG. 12. When composition 2 was used during process step 1 thehydrodynamic radii already after five freeze and thaw cycles wereremarkably increased in conjunction with the formation of higher orderaggregates and were not measurable after ten freeze and thaw cycles andwhen used in process step 2 due to further increased radii and higherorder aggregates which were outside the DSL measure limit (FIG. 13 C andD). The behavior of the adenoviral particle size in composition 1 and 2prepared either during process step 1 and 2 after the application offive freeze and thaw cycle is depicted in FIG. 13 A to D).

In summary and conclusion, composition 1 generally exhibited excellentstabilizing efficacy for the adenoviral vector particles during bothapplied early production steps. In contrast, although composition 2showed stabilizing efficacy when used directly afterultracentrifugation, reduced stabilizing efficacy was observed when usedlater in the production process as compared to composition 1.

The DLS data correlate with the in vitro infectivity data shown inexample 1. This leads to the conclusion that the use of specificallytailored stabilizing compositions based on amino acids early in theproduction process of viral vector compositions is important for thestability during further processing steps in biopharmaceuticalmanufacturing. Moreover, the stabilization of viral vector basedcompositions in terms of the decrease of the polydispersity of thesolution results in solutions with high in vitro infectivity.

EXAMPLE 3

In vitro study of the functional and structural integrity of adenoviralvectors during liquid storage at 25° C. and at 37° C. showed that aminoacid based compositions comprising at least three, four and or fiveexcipients, preferably amino acids in combination with a sugar, e.g.sucrose in a ratio of amino acids to sugar of at least 1:2, canremarkably retain infectivity of the viral vectors in cell culture andretain the particle size distribution with polydispersity index valuesbelow 0.3.

3.1 Materials and Methods

Compositions 3, 4, 5 and 6 contained the following 3 amino acids:

-   -   histidine, glutamic acid, methionine (composition 3),    -   histidine, lysine, methionine (composition 4),    -   histidine, glycine, methionine (composition 5) and    -   histidine, alanine, glutamic acid (composition 6), respectively.

Composition 7 and 8 contained the following 4 amino acids:

-   -   histidine, lysine, glycine, arginine (composition 7), and    -   histidine, lysine, alanine, methionine (composition 8),        respectively.

Composition 9 contained the 5 amino acids histidine, glycine, alanine,glutamic acid and methionine.

All compositions additionally contained 40 g/1 saccharose and 2 mM MgCl₂in a fixed concentration resulting in different amino acid to sugarratios of 1:3; 1:1.3; 1:1.6 and 1:1.5 in the case of compositions 3, 4,5 and 6, respectively. In the case of compositions 7 and 8 the aminoacid to sugar ratio was 1.1:1 in both compositions. In composition 9 theamino acid to sugar ratio was adjusted to 1:1.5. The pH value wasadjusted in all compositions to 7.4.

An adenoviral stock solution stored at −80° C. with a concentration of1*1011 IFU/ml in the original supplier formulation (Firma Sidon;Martinsried/Munich; Germany) was employed. The original supplierformulation contained 10 mM HEPES, pH 8, 4 g/I saccharose and 2 mMMgCl₂.

3.1.1 Sample Preparation and Liquid Storage

HEK293 cells were transduced with high titres of adenovirus 5 vectorscontaining the coding DNA for the eGFP protein. 48 h after transduction,cells were harvested and the release of viral particles was performedwere released via Na-Deoxycholat and DNase I treatment. Viral particleswere purified and concentrated by CsCI gradient ultracentrifugation,followed by buffer exchange in the original supplier formulation on PD10columns and subsequent determination of the infective titre. Theresulting high titre adenoviral stocks were subsequently aliquoted andstored at −80° C. The initial titre of the adenoviral stock solutions inthe original supplier formulation was determined to be about 2*10¹¹IFU/ml. The adenoviral vector stock solution was re-buffered by dilutionto a concentration of 5*10⁸ IFU /ml in stock solutions comprising basecomponents of the compositions according to paragraph section 3.1 aboveand was subsequently further diluted into the final sample concentrationof 1*10⁸ IFU/ml using 1.25× concentrates of compositions according toparagraph section 3.1 above, resulting in the adenoviral vectorformulations for subsequent liquid storage. 50 μl of the adenoviralvector formulations were aliquoted in sterile 100 μPCR vials andsubsequently stored for up to 3 months at 5° C. as well as 25° C. andfor up to 35 days at 37° C., respectively. At the indicated time pointsduring liquid storage as well as at the initial time point t=0, theinfective titres were determined by virus titration in HEK 293 cellcultures according as detailed in to paragraph section 1.1.2 above wasdetermined. In parallel, the hydrodynamic radii of the adenoviralparticles and the corresponding polydispersity indices were measured byDLS according as described in section to paragraph 1.1.3 above using aslightly different protocol as follows. The DLS measurements wereperformed with 10 μl of adenoviral vector preparations diluted withsterile filtrated water (0.02 nm) to suitable concentrations of theadenoviral vector and with acquisition times between 1 and 3 seconds in80 and 40 cycles, respectively. The resulting autocorrelation functionswere analysed using the DynaPro DLS software resulting in the evaluationof the hydrodynamic radii (nm) as well as the particle size distributionwith respect to the polydispersity indices (PDI) Special ingredients ofthe compositions according to the invention contributed to the resultingautocorrelation functions of the analyzed adenovirus particles. Thus,differences of the resulting autocorrelation functions and thecompositions without viral vectors led to the calculated results of theDLS measurements.

Electron microscopic analysis was performed as described in paragraph1.1.4 above.

3.2. Results Liquid Storage

Liquid storage for 3 months at 5° C. in compositions 3 to 9 according tothe invention (paragraph see section 3.1 above) comprising three, fourand or five amino acids revealed the complete retention of the infectivetitre of about 1×10⁸ IFU/ml compared to the positive control (FIG. 19A).In contrast, the infective titre of the adenoviral particles stored inthe original supplier formulation was remarkably reduced to approx.1×10⁶ IFU/ml after liquid storage for three months at 5° C. (FIG. 19A).Moreover, liquid storage of the adenoviral vector particles in theoriginal supplier formulation at 25° C. resulted already after liquidstorage for 21 days in a reduction of the infective titre to about 1×10⁶IFU/ml after liquid storage for 21 days and after liquid storage for 1month to a further the reduction in the infective titre was furtherreduced to about 1×10⁵ IFU/ml after liquid storage for 1 month. Furtherliquid storage for 3 months at 25° C. in the original supplierformulation resulted in the complete loss of the infective titer of theadenoviral particles (FIG. 19B). On the other hand, formulation of theadenoviral particles in compositions 3 to 9 comprising three, four andor five amino acids, respectively, resulted in the nearly completeretention of the infective titres of the adenoviral vector preparationseven after liquid storage for 3 months at 25° C. (approx. 1×10⁷ IFU/ml;FIG. 19B). Even liquid storage for 21 days at 37° C. led to a remarkableretention of the infective titre of the adenoviral vector particlesformulated in composition 4 and 8 comprising three and or four aminoacids, with a residual titre of about 1×10⁶ IFU/ml compared to thecomplete loss of the infective titre in the original supplierformulation during liquid storage for 21 days at 37° C. While furtherliquid storage for 35 days at 37° C. led to the loss of infective titrein composition 4, the corresponding titre of the adenoviral vectors wasretained to about 1×10⁵ IFU/ml in composition 8 comprising four aminoacids (FIG. 19C).

DLS measurement

The molecular integrity of the adenoviral vector compositions duringliquid storage for 14 days to about 35 days at 37° C. was analyzed usingDLS measurements. In addition to the evaluation of the hydrodynamicradii of the adenoviral particles, the polydispersity indices (PDI) aswell as the values for D10, D50 and D90 as parameters for the particlesize distribution in the adenoviral particle compositions weredetermined. In FIG. 15 shows the PDI values of the adenoviral vectorsformulated in composition 6, 8 and 9 in comparison to the correspondingPDI values in the original supplier formulation after liquid storage for14 days and 35 days at 35° C. are depicted. The initial PDI value attime point t=0 of the adenoviral vector compositions formulated in theoriginal supplier formulation was already remarkably increased (PDIapprox. 0.25) associated with a strong standard deviation as compared tothe corresponding PDI values in compositions 6, 8 and 9. These findingssuggesting the appearance of big particles with fluctuations in size inthe original supplier formulation as compared to the adenoviralparticles in compositions 6, 8 and 9 with a narrow particle sizedistribution at time point t =0 (PDI around 0.1). After 14 days ofstorage at 35° C., the particle size distribution increased incompositions 6, 8 and 9 to different extent slightly but remainedbetween the values of 0.1 and 0.2. In the original supplier formulation,the particle size distribution slightly decreased after liquid storagefor 14 days at 37° C. After liquid storage for 35 days at 37° C. the PDIvalues further increased in all formulations, particularly in theoriginal supplier formulation, where it increased to a PDI value ofapprox. 0.26 associated with a large standard deviation suggesting theappearance of big particles with variable size. In compositions 6 and 8,the particle size distribution corresponding to the PDI values was alsoincreased but to a minor extent compared to the original supplierformulation, namely in the case of composition 6, the PDI was 0.234 andin the case of composition 8, the PDI was 0.171. The large standarddeviation in the original supplier formulation and the remarkableincrease in PDI may explain the loss of infectivity in this formulationafter liquid storage for 35 days at 37° C. as a result of the appearanceof big particles of variable sizes. In contrast, liquid storage of theadenoviral vectors in composition 8 comprising four amino acids resultedin the retention of a PDI value of 0.171 with a small standard deviationsuggesting that the appearance of the majority of the measured particlesrepresents infective particles associated with a narrow particle sizedistribution (FIG. 19D).

Transmission Electron Microscopy

In addition, the molecular integrity of the adenoviral vectorpreparations formulated in compositions 5 and 8 after liquid storage for2 months at 25° C. was further analyzed using transmission electronmicroscopy as described in paragraph section 1.1.4 above. In theacquired electron microscopic images the majority of the adenoviralparticles were observed as intact icosahedral shaped bright particles(FIG. 20 and 21; black arrows). The diameter of the adenoviral particleswas measured to be approx. 100 nm from vertex-to-vertex. The adenoviralparticles appeared preferentially as single entities. Nevertheless, thebackground showed the presence of adenoviral vector debris such as largepleomorphous structures and smaller granular structures, possiblyrepresenting adenoviral subcomponents such as hexons and fibers.Occasionally, aggregation of adenoviral particles and debris could beobserved (FIGS. 20 and 21).

EXAMPLE 4

Analysis of the infective titre of adenoviral vector compositions at theindicated time points during liquid storage at 37° C. revealed a betterstabilizing efficacy of a composition comprising 4 amino acids accordingto the invention as compared to an original supplier formulation.

4.1 Materials and Methods

Composition 11 used in this example contained the four amino acidshistidine, lysine, alanine, methionine in combination with 40 g/Isaccharose resulting in an amino acid to sugar ratio of 1.1:1. The pHvalue of the formulation was adjusted to 7.4. For comparison, a standardoriginal supplier formulation 2 comprising 1.522 g/I histidine, 50 g/Isaccharose, 1 mM MgCl₂, 1.211 g/I Tris, 4.383 g/l NaCl, 0.029 g/l EDTA,0.005% (v/v) ethanol and 0.2% polysorbat 80 at a pH of 7.4 was applied.

An adenovirus serotype 5 (Ad5) stock solution stored at −80° C. with aconcentration of 2×10¹¹ IFU/ml in the original supplier formulation(Sirion; Martinsried/Munich; Germany) was employed. The originalsupplier formulation contained 10 mM HEPES, pH 8, 4 g/I saccharose and 2mM MgCl₂.

4.1.1 Sample Preparation and Liquid Storage

HEK293 cells were transduced with high titres of adenovirus 5 vectorscontaining the coding DNA for the eGFP protein. 48 h after transduction,cells were harvested and viral particles were released viaNa-Deoxycholat and DNase I treatment. Viral particles were purified andconcentrated by CsCI gradient ultracentrifugation, followed by bufferexchange in the original supplier formulation on PD10 columns andsubsequent determination of the infective titre. The resulting hightitre adenoviral stocks were subsequently aliquoted and stored at −80°C. The initial titre of the adenoviral stock solutions in the originalsupplier formulation was determined to about 2*10¹¹ IFU/ml. In the firststep the adenoviral stock solution with an initial infectivity of 2×10¹¹IFU/ml was 1:2 diluted with the original supplier formulation to get astarting concentration of 1×10¹¹ IFU/ml. The adenoviral vector stocksolution was re-buffered by 1/200 dilution to a concentration of 5*10⁸IFU /ml in stock solutions comprising base components of thecompositions according to section 4.1 above. Subsequently, samples werefurther 1:5 diluted with 1.25× concentrates of compositions according tosection 4.1 above, to reach the final sample concentration of 1*10⁸IFU/ml for subsequent liquid storage. 50 μl of the adenoviral vectorformulations were aliquoted in sterile 100 μl PCR vials and subsequentlystored for up to 21 days at 37° C. Infective titres according to section1.1.2 above were determined by virus titration in HEK293 cell culturesat the initial time point t=0 and at the indicated time points duringliquid storage.

4.2. Results Liquid Storage

Liquid storage of the adenoviral vectors formulated in composition 11comprising four amino acids resulted in a better retention of theinfective titre after liquid storage for 14 days at 37° C. and morepronounced after liquid storage for 21 days at 37° C. compared to thestandard original supplier formulation 2 (FIG. 22). This data furtherconfirmed the results of example 3 above, in which the most effectivestabilizing composition also comprised the four amino acids histidine,lysine, alanine, methionine in combination with 40 g/I saccharose in anamino acid to sugar ratio of 1.1:1 (composition 8). Therefore, thecombination of the four amino acids histidine, lysine, alanine,methionine in combination with 40 g/I saccharose and an amino acid tosugar ratio of 1.1:1 exhibited superior stabilization of adenoviralvector particles over the standard formulation as described in section4.1, during thermal stress and liquid storage.

EXAMPLE 5

In vitro study of the functional and structural integrity of adenoviralvectors after different processing steps and subsequent application ofseveral freeze-thaw cycles as well as during liquid storage at 37° C.showed that amino acid based compositions comprising at least three,four and five excipients preferably amino acids in combination with asugar, e.g. sucrose in a ratio amino acids to sugar of at least 1:2remarkably retained infectivity of the viral vectors in cell culture andretained the particle size distribution with polydispersity index valuesbelow 0.3.

5.1 Materials and Methods

Composition 11 contained the three amino acids alanine, histidine,glutamic acid in combination with 40 g/I saccharose in an amino acid tosugar ratio of 1:1.5. Composition 12 used in this example contained thefour amino acids histidine, lysine, alanine, methionine in combinationwith 40 g/I saccharose resulting in an amino acid to sugar ratio of1.1:1. The pH values of the formulations were adjusted to 7.4. Forcomparison, a standard original supplier formulation 2 comprising 1.522g/I histidine, 50 g/l saccharose, 1 mM MgCl₂, 1.211 g/I Tris, 4.383 g/lNaCl, 0.029 g/l EDTA, 0.005% (v/v) ethanol and 0.2% polysorbat 80 at apH of 7.4 and another standard original supplier formulation 1comprising 10 mM HEPES, pH 8, 4 g/l saccharose and 2 mM MgCl₂ as well asthe standard buffer PBS were applied.

An adenovirus serotype 5 (Ad5) stock solution stored at −80° C. with aconcentration of 2×10¹¹ IFU/ml in the original supplier formulation(Sirion; Martinsried/Munich; Germany) was employed. The originalsupplier formulation contained 10 mM HEPES, pH 8, 4 g/I saccharose and 2mM MgCl₂.

A Modified Vaccinia Ankara (MVA) viral vector in Tris-HCL (pH 9) wasused for freeze-and-thaw experiments and subsequent DLS analyses.

5.1.1 Sample Preparation and Liquid Storage

HEK293 cells were transduced with high titres of adenovirus 5 vectorscontaining the coding DNA for the eGFP protein. 48 h after transduction,cells were harvested and the release of viral particles was performedvia Na-Deoxycholat and DNase I treatment. Viral particles were purifiedand concentrated by CsCI gradient ultracentrifugation. The furtherformulation of the adenoviral vector particles was performed in thefollowing two different processing steps.

Processing step 1: Adenoviral vector formulations were prepared byre-buffering of the adenoviral vector preparations immediately afterCsCI gradient ultracentrifugation. In a first step the obtainedconcentrated and harvested adenoviral vector band was diluted 1 per 1 inthe standard original supplier formulation 1 and re-buffered usingdialysis at 2-8° C. in compositions 11 and 12 according to the invention(as described in 5.1) as well as in the standard original supplierformulations 1 and 2 and in the standard buffer PBS.

Processing step 2: After ultracentrifugation, the obtained concentratedand harvested adenoviral vector band was diluted 1:1 in the standardoriginal supplier formulation 1 and re-buffered using dialysis at 2-8°C. in the standard original supplier formulation 1. The resulting hightitre adenoviral stocks were subsequently aliquoted and stored at −80°C. After thawing the adenoviral vectors in the standard originalsupplier formulation 1 were re-buffered using dialysis at 2-8° C. for asecond time in composition 11 according to the invention (described inparagraph 5.1) and in the standard buffer PBS.

The initial titre of the adenoviral stock solutions in the standardoriginal supplier formulation 3 and 1 as well as in the compositionsaccording to the invention after the both processing steps wasdetermined to be about 1*10¹¹ IFU/ml. For the subsequent application ofseveral freeze and thaw cycles 50 μl of the high titre adenoviral vectorformulations were aliquoted in sterile 100 μl PCR vials and subjected torepeated freeze (1 h at −80° C.) and thaw (1 h at room temperature)cycles. At time point t=0 and after the application of 5, 10, 15 and 20freeze and thaw cycles the infective titres was determined by virustitration in HEK 293 cell cultures according to paragraph 1.1.2. Inparallel, the hydrodynamic radii of the adenoviral particles and thecorresponding polydispersity indices were measured by DLS according toparagraph 1.1.3 using a slightly different protocol according toparagraph 3.1.1.

For subsequent liquid storage at 37° C. the high titre adenoviral stocksolutions in the different formulations were further diluted to aninfective titre of around 1*10⁸ IFU/ml. 50 μl of the diluted adenoviralvector formulations were aliquoted in sterile 100 μl PCR vials andsubsequently stored for up to 28 days at 37° C. At the initial timepoint t=0 and after 14 days and 28 days of liquid storage at 37° C. theinfective titres was determined by virus titration in HEK 293 cellcultures according to paragraph 1.1.2. In parallel, the hydrodynamicradii of the adenoviral particles and the corresponding polydispersityindices were measured by DLS according to paragraph 1.1.3 using aslightly different protocol according to paragraph 3.1.1.

For MVA formulation, composition 13 comprising three amino acids,histidine, methionine, alanine, was used. Subsequently, freeze-thawcycles were applied and samples were analyzed by DLS.

5.2. Results Liquid Storage

In FIG. 23, the in vitro infectivity of the adenoviral preparationsafter the two different processing steps (PS) during liquid storage forup to 28 days at 37° C. is depicted. Formulation of the adenoviralvectors in composition 12 and 11 showed a remarkable higher retention ofthe infective titre prepared by processing step 1 (PS1) during liquidstorage at 37° C. compared to the standard buffer PBS and maintainedcomparable to the two standard original supplier formulations 2 and 1.Preparation of the adenoviral vector formulations according toprocessing step 2 (PS2) showed higher stabilization of the infectivetitre of the adenoviral vectors formulated in compositions 11 comparedto the formulation in the standard buffer PBS and even compared to theadenoviral vector preparation in the original supplier formulationprepared by processing step (PS1) during liquid storage for 28 days at37° C.

In vitro infectivity after the application of several freeze and thawcycles

Formulation of the adenoviral vector preparations prepared according toprocessing step 1 (PS1) in compositions 11 and 12 showed a remarkablemaintenance of the in vitro infectivity, particularly after theapplication of 15 freeze and thaw cycles compared to the originalsupplier formulations 2 and 1 (FIG. 24A). In contrast, formulation ofthe adenoviral vector preparations in the standard buffer PBS resultedalready after the processing step 1 (PS 1) in the complete loss of theinfective titre (FIG. 24A). Moreover, formulation of the adenoviralvector preparations according to processing step 2 in composition 11showed a nearly complete retention of the in vitro infectivity afterapplication of up to 10 freeze and thaw cycles (FIG. 20 B). In contrast,formulation of the adenoviral vector preparations in the standard bufferPBS according to processing step 2 resulted already after theapplication of 5 freeze and thaw cycles in the complete loss ofinfectivity (FIG. 24B).

DLS Measurement

Similar observations were made in the parallel performed DLSmeasurements. Sample preparation according to processing step 1 (PS 1)resulted in the nearly complete retention of the particle sizedistribution in the adenoviral vector preparations directly after samplepreparation (0) expressed in the calculated polydispersity indicessmaller 0.3 in the compositions 11 and 12 as well as in the originalsupplier formulations 1. In contrast, formulation of the adenoviralvector preparations in the standard buffer PBS according to processingstep 1 (PS1) showed a higher particle size distribution withpolydispersity indices>0.3 already after the preparation and morepronounced after the application of only 5 freeze and thaw cycles. Thefurther application of 10 and 20 freeze and thaw cycles resulted in thecomplete degradation of the viral particles in formulated PBS. Thefurther application of 5, 10 and 20 freeze and thaw cycles to theadenoviral vector preparation formulated in composition 11 and 12according to the invention led to the nearly complete retention of theinitial particle size distribution<0.3. In contrast, formulation of theadenoviral vector preparations in the original supplier formulation 1resulted in a remarkably increased PDI>0.3 after the application of 10as well as 20 freeze and thaw cycles associated with increasing standarddeviations, suggesting the appearance of bigger particles with variablesize in addition to the main adenoviral vector particles (FIG. 25).

Sample preparation according to processing step 2 (PS 2) revealed theretention of the particle size distribution (PDI<0.3) after applicationof 5, 10, 15 and 20 freeze and thaw cycles in the case of the adenoviralvector preparations formulated in composition 11 and 12 according to theinvention. Contrary, the formulation of the adenoviral vectors in thestandard buffer PBS according to processing step 2 (PS 2) resulted inthe complete loss of intact adenoviral particles after the applicationof 10, 15 and 20 freeze and thaw cycles.

Similar experiments were performed with MVA in composition 13 comparedto the original supplier formulations 2 and 1 and PBS (FIG. 26). WhenMVA was formulated in composition 13, the PDI was smaller than 0.5. Incontrast, in the original supplier formulations 1 and 2 already aftersample preparation and after the application of 20 freeze-thaw cyclesshowed PDI values higher than 0.5.

1-15. (canceled)
 16. A method for preparing liquid compositionscomprising modified viruses for storage, wherein the modified virusespresent in the composition are used to deliver genetic material intocells, the method comprising the steps: (a) providing modified viruses;(b) providing a solution comprising at least one sugar and at leastthree different amino acids, wherein the three different amino acids areselected from at least three different groups from the following: aminoacids with a polar functional group, amino acids with an aliphaticfunctional group, amino acids with an aromatic functional group, aminoacids with a negatively charged functional group, and amino acids with apositively charged functional group; and wherein the solution comprisesan amino acid-sugar ratio of no more than twice the amount of sugar ascompared to the amount of amino acids; and (c) mixing the modifiedviruses of step (a) with the solution of step (b) to form a mixture; and(d) storing the mixture of step (c) as a liquid; and wherein the methoddoes not comprise drying the mixture.
 17. The method of claim 16,wherein storing the mixture in step (d) comprises storing for at least28 days.
 18. The method of claim 16, wherein the viruses are attenuatedviruses.
 19. The method of claim 18, wherein the viruses have beenattenuated to be replication deficient.
 20. The method of claim 19,wherein the viruses can infect target cells.
 21. The method according toclaim 19, wherein the viruses are from modified vaccinia ankara (MVA)viruses, modified adenoviruses, modified Adenovirus-associated viruses(AAV), modified lentiviruses, modified vesicular stomatitis viruses(VSV), or modified herpesviruses.
 22. The method according to claim 19,wherein the viruses are in the form of virus like particles.
 23. Themethod according to claim 19, further comprising adding an antigenicpolypeptide.
 24. The method according to claim 19, further comprisingadding at least one adjuvant.
 25. The method according to claim 19,wherein the viruses are replication-deficient viral viruses that havebeen reconstituted immediately after harvesting from cell cultures andpurification.
 26. A virus composition obtained or obtainable by themethod according to claim
 16. 27. The virus composition of claim 26 foruse as a prime-boost vaccine.
 28. The virus composition according toclaim 26, wherein the virus composition is for intramuscular,subcutaneous, intradermal, transdermal, oral, peroral, nasal, and/orinhalative application.